Epidemiology of rotavirus-norovirus co-infection and determination of norovirus genogrouping among children with acute gastroenteritis in Tehran, Iran

Background: Enteric viruses, particularly human rotavirus and norovirus, have been shown to replace bacteria and parasites, as the most common pathogens responsible for acute diarrhea. However, there are still few epidemiological data on the simultaneous occurrence of these viruses in Iran. In this regard, the aim of this study was to assess the useful epidemiological data on the gastroenteritis associated with rotavirus-norovirus mixed infection and to examine the prevalence of norovirus genogrouping among children aged less than five years old in Iran

Methods: A total of 170 stool samples were collected from children under five years of age with the clinical signs and symptoms of acute gastroenteritis, from May 2013 to May 2014. For the detection of both rotavirus and norovirus, total RNA was extracted from all samples, followed by reverse transcription polymerase chain reaction [RT-PCR]. For both detected rotaviruses and noroviruses, genogrouping was performed

Results: Of 170 samples, 49 [28.8%] and 15 [8.8%] samples were found to be positive for rotavirus and norovirus infections by RT-PCR. Interestingly, 6 [3.5%] patients were positive for both infections. Among the 15 norovirus-positive patients, 13 [86.6%] and 2 [13.3%] belonged to genogroups GII and GI

Conclusions: The norovirus genogroup GII and rotavirus lead to the serious infections in children with acute gastroenteritis. However, more well-designed studies are needed to further elucidate the role of other enteric viruses in acute gastroenteritis

Construction of a minigenome rescue system for measles virus, AIK-c strain

In the recent decade, the reverse genetics method has been broadly used for rescue of negative-stranded RNA viruses from cDNA or viral minigenomes. This technique has been applied to study different steps in virus replication and virus-host interactions. Reverse genetics could also be implemented for design of new vaccines. The T7 RNA polymerase activity as well as virus [nucleocapsid protein] N, [phosphoprotein] P and [Large] L proteins are necessary to rescue the virus or viral minigenome. Measles virus is a negative-stranded non-segmented RNA virus. There are useful vaccine strains to prevent measles disease. Here, we describe the construction of a new helper cell line for rescue of measles virus minigenome. The helper cell line stably expresses T7 RNA polymerase as well as measles virus N and P proteins by tricistronic mRNA. Materials and Methods: For rescue of measles virus minigenome a stable helper cell line by using tricistronic expression vector was developed which expressed T7 RNA polymerase as well as measles virus N and P proteins. To construct the tricistronic expression vector, T7 RNA polymerase gene was cloned after cytomegalovirus [CMV] promoter and measles virus N and P proteins were under control of IRES [internal ribosome entry site] sequences. Our results indicated that measles virus minigenome could be rescued in this constructed helper cell line. Through this system, the measles virus minigenome was rescued. Further studies are necessary to improve the rescue efficiency. This may be possible by replacing the CMV promoter with the T7 promoter

Construction of an eGFP expression plasmid under control of T7 promoter and IRES sequence for assay of T7 RNA polymerase activity in mammalian cell lines

Recently, the use of T7 RNA polymerase instead of other viral and cellular promoters is increasing due to high efficacy of transcription in the cell cytoplasm by this polymerase. In order to translate the transcripts produced by T7 RNA polymerase in mammalian cell lines, it is necessary to include Internal Ribosome Entry Site [IRES] sequences. In addition, if sequence of poly A signal would be included after interested gene, the rate of expression could be increased in the cells. For expression of eGFP in HEK-293 and T7-BHK cells by T7 RNA polymerase, the sequence of eGFP as well as IRES sequences upstream of eGFP gene and poly A signal were inserted into a pUC57 plasmid. On the other hand, gene of T7 RNA polymerase was cloned into modified pIRES2-EGFP plasmid. Then, the constructed plasmids were transfected into HEK-293 cells. T7-BHK cell was used for control of T7 RNA polymerase activity. Our results showed that using T7 RNA polymerase for expression of foreign genes in mammalian cell lines is highly efficient. Highly efficient eGFP expression in HEK-293 cells showed that T7 RNA polymerase could be used for cytoplasmic RNA transcription such as production of anti-cancer proteins and oncolytic viral genomic RNA by reverse genetics

[Transfection of a hepatoma cell line by cloned HBV DNA and evaluation of transcription and expression of viral-specific markers and interferon stimulated genes]

Despite availability of an effective vaccine against hepatitis B virus [HBV], the global prevalence of this virus infection has not diminished significantly. Contrary to numerous other human viruses, HBV does not have the ability to propagate in cell culture. However, infectious virus has been produced by transfection of human hepatoma cells with plasmids that contain full length HBV genome. Generation and optimization of appropriate cell culture systems can help us in demonstrating the quality of genome replication by PCR as well as expression of surface antigen secretion. Interferon stimulating genes [ISGs] are usually produced in response to interferon and can be determined as a measure of response to IFN-therapy. Therefore, in pharmacological studies, in addition to assessing the effects of a medicine on viral determinants of replication, its' effects on stimulation of various ISGs, as indicators of innate immune responses, can be achieved. In this study, we transfected the Huh-7 hepatoma cell line with pCH-9/3091. HBsAg production and viral mRNA transcription were subsequently evaluated. In this system, by using ISGs-specific primers, the ISG mRNAs recognition method was optimized and utilized. Huh-7 cells supported HBV replication. The peak HBsAg secretion was observed at 72 h post-transfection. By using designed primers for the S and pg/pC regions, transcription and genome replication of the virus was shown. RT-PCR results for ISG production by transfected cells showed no role for HBV in enhancement of ISGs levels in Huh-7 cells. The results indicated that this system can be used for functional studies of HBV-specific genes as well as assessment of the effects of new drugs or new vaccines. In addition, it may be used to study the mechanisms of drug resistance that have resulted in difficulties in response to HBV antivirals, including IFN-alpha

[Detection of HCMV infection in glioma brain tumors]

Human cytomegalovirus [HCMV] is a beta-herpesvirus that causes persistent infection in humans, as well as severe disease in fetuses and immunocompromised individuals. Although HCMV is not currently causally implicated in human cancer, emerging evidence suggests that HCMV infection may be specifically associated with malignancies such as gliomas. Gliomas are one of the most common brain tumors that affect humans. It is classified into four grades. In this study, we have developed and used a real-time PCR method for the detection and diagnosis of HCMV infection in glioma brain tumor samples. Paraffin-embedded tumor samples were chosen from patients who referred to Imam Khomeini Hospital Neurosurgery Ward. DNA was extracted from paraffinembedded tissues by a DNA extraction kit. After designing specific primers for the HCMV US28 region, a real-time PCR method was developed for detection of HCMV US28. The results of qualitative real-time PCR on 4/18 patients [22.2%] were positive. Two patients with positive HCMV results died. This is the first study that has monitored HCMV genes in samples from glioma patients in Iran. Considering the results of this study and controversies associated with other studies, a more comprehensive study using this and other diagnostic methods is suggested.

Immune responses against a new HIV-1 p24-gp41/pCAGGS-IL-12 DNA vaccine in balb/c mice

Development of an effective vaccine is highly needed in order to restrict the AIDS pandemic. DNA vaccines initiate both arms of immunity without the potential of causing disease. HIV-1 p24 and gp41 [gag and env] proteins play important roles in viral pathogenesis and are effective candidates for immune induction and vaccine design. In this study, new DNA vaccine candidates constructed from HIV-1 fused p24- gp41 or gp41 alone were evaluated in Balb/c mice for induction of cellular and humoral immune responses. Recombinant plasmids, pcDNA3.1/Hygro expression vector containing immunogenic sequences of fused p24-gp41 or gp41alone were produced. Dendrosome used as a system for carrying vectors in laboratory animals, and an IL-12 containing vector [pCAGGS-IL-12] was co-immunized with the p24-gp41 vector as a genetic adjuvant. Induction of effective immune responses against the designed vectors as DNA vaccine candidates in Balb/c mice was evaluated. Levels of total antibodies, IgG isotypes [IgG2a and IgG1]? IFN-alpha and IL-4 were measured by ELISA. MTT assay was used to evaluate lymphoproliferation. The results confirmed that the immunogenic epitopes of both p24 and gp41 genes are highly effective inducers of immune responses, and administration of fused p24-gp41 alone or along with IL-12 resulted in further enhancement of immune responses. Group 4 that received fused fragments [p24-gp41] along with an IL-12 expressing vector demonstrated a significantly higher Stimulation Index [SI] and IFN-alpha production [p<0.0001] with a significant increase in IgG2a/IgG1 ratio, indicating the stimulation of CMI towards Th1. Although gp41 containing vector [group 6] also showed significant increases in both proliferation and IFN-alpha production, the responses were persistently lower than that of p24-gp41 containing vectors. Total antibody production was highest in group 6 as expected. Dendrosome proved to be an efficient carrier of recombinant plasmids constructed in this study. Further studies are necessary to evaluate these constructs as HIV vaccine candidates

[Cloning and evaluation of expression of a novel overlapping region of NS3 gene of Hepatitis C virus by expressing vector]

In this project, our aim was to construct a novel expressing vector harboring a new sequence from overlapping region of NS3 gene of HCV from infected Iranian patient. The partial NS3 [pNS3] gene was amplified by Nested-RT-PCR method using sera of HCV infected patients harboring genotype 1a. After purification and cloning the pNS3 into TA-cloning vector, the best colony was selected based on Blue/White screening and colony-PCR following by confirmation with sequencing and restriction digestion with BglII. The sequenced gene was compared with other reference sequences using alignment softwares. The resultant pNS3 gene subcloned into the expression vector, IRES vector, followed by selection the suitable clones by 2 different colony-PCRs. The gene expression was evaluated using GFP detection, RT-PCR and western blotting techniques after transfection of the IRES-pNS3 vector into the 293 cell line. After pNS3 sequence amplification by RT-PCR, sequencing results showed high homology among the sequences with other reference sequences. This result also showed that it belonged to genotype 1 of HCV. Colony-PCR showed the insertion of gene into expressing vector with the right orientation. GFP expression, RT-PCR and western blotting confirmed transfection of vector, expression of pNS3 gene and production of its protein in 293 cells respectively. This novel expressing vector harboring partial region of NS3 gene in compare to full NS3 gene maybe more useful in immune induction by antigen presenting cells due to absence of genes responsible for protease activity of the protein in the setting of HCV vaccine

[Construction of a bicistronic eukaryotic vector expressing M1 and NP genes of influenza virus]

In this study, two conserved genes [M1 and NP] of influenza virus were expressed in a bicistronic vector in order to develop a universal gene based vaccine. Plasmids M1-pIRES2-EGFP, pIRES2-NP were constructed by cloning the PCR products of M1 and NP genes which were amplified from the A/Peurto Rico/8/34 [H1N1] influenza virus strain into the plasmid expression vector pIRES2-EGFP, respectively. For construction of M1-pIRES2-NP bicistronic plasmid, M1 gene was extracted from M1-pIRES-EGFP plasmid and subcloned into pIRES2-NP construct. Finally, simultaneous expression of both genes was assessed by transient transfection of bicistronic plasmid into BHK-21 cell lines and subsequent immunofluorescence staining. The results of enzymatic double digestions on the constructed plasmids and sequencing demonstrated the success of cloning processes of above mentioned genes. Correct expression of these genes was confirmed by M1-pIRES2-NP plasmid expression in BHK-21 cell lines confirmed by immunofluoresence microscopy. Simultaneous expression of influenza M1 and NP genes from a bicistronic plasmid containing "IRES" sequence is achievable

Rapid low-cost detection of hepatitis C virus RNA in HCV-infected patients by real-time RT-PCR using SYBR green I

We intend to design and validate a low-cost assay for the detection of hepatitis C virus [HCV] RNA using rapid-cycle RT-PCR. The procedure is performed in a closed system with little risk of contamination allowing PCR and product identification to be performed within one or two hours. A SYBR Green-based real-time RT-PCR for rapid detection of HCV. Amplicon synthesis was monitored continuously by SYBR Green I, which binds to double stranded DMA during PCR. The PCR products were identified by melting curve analysis. Standard sera with known concentrations of HCV RNA and 150 clinical samples were used to validate our assay. The minimum detection level of our assay was less than 50 ID/mL. The results on 100 plasma samples were comparable with commercial assays. This method is useful for rapid qualitative detection of HCV infection and particularly suitable for routine diagnostic applications

Prediction of hepatitis B virus lamivudine resistance based on YMDD sequence data using an artificialneural network model

Hepatitis B virus [HBV] infection is an important health problem worldwide with critical outcomes. The nucleoside analog lamivudine [LMV] is a potent inhibitor of HBV polymerase and impedes HBV replication in patients with chronic hepatitis B. Treatment with LMV for long periods causes the appearance and reproduction of drug-resistant strains, rising to more than 40% after 2 years and to over 50% and 70% after 3 and 4 years, respectively. Artificial neural networks [ANNs] were used to make predictions with regard to resistance phenotypes using biochemical and biophysical features of the YMDD sequence. The study population comprised patients who were intended for surgery in various hospitals in Tehran-Iran. An ACRS-PCR method was performed to distinguish mutations in the YMDD motif of HBV polymerase. In the training and testing stages, these parameters were used to identify the most promising optimal network. The ideal values of RMSE and MAE are zero, and a value near zero indicates better performance. The selection was performed using statistical accuracy measures, such as root mean square error [RMSE], coefficient of determination [R2], and mean absolute error [MAE]. The main purpose of this paper was to develop a new method based on ANNs to simulate HBV drug resistance using the physiochemical properties of the YMDD motif and compare its results with multiple regression models. The results of the MLP in the training stage were 0.8834, 0.07, and 0.09 and 0.8465, 0.160.04 in the testing stage; for the total data, the values were 0.8549, 0.115, and 0.065, respectively. The MLP model predicts lamivudine resistance in HBV better than the MLR model. The ANN model can be used as an alternative method of predicting the outcome of HBV therapy. In a case study, the proposed model showed vigorous clusterization of predicted and observed drug responses. The current study was designed to develop an algorithm for predicting drug resistance using chemiophysical data with artificially created neural networks. To this end, an intelligent and multidisciplinary program should be developed on the basis of the information to be gained on the essentials of different applications by similar investigations. This program will help design expert neural network architectures for each application automatically

[Evaluation of intramuscular versus intra-dermal inoculation of bacterial plasmid encoding nucleoprotein of influenza virus]

The use of bacterial plasmids carrying specific genes of pathogens as genetic vaccines is a relatively new technique for induction of cellular immune responses against microbial pathogens. Mechanisms of production of specific immune responses against these vaccines are not still completely understood. Therefore, it is necessary to examine various routes of inoculation to find the best way of immunization for specific antigens. In this research, intramuscular method of inoculation of influenza vaccine nucleoprotein [NP] encoding vector was compared with that of intra-dermal method. In this study, the ability of two different methods of immunization [intramuscular and intra-dermal] in induction of CTL responses as well as their efficiency in clearance of influenza virus from the lung of BALB/c mice was compared. Female BALB/c mice were immunized with influenza virus NP expressing plasmids on days 0, 14 and 28. CTL activity of mice was evaluated by lactate dehydrogenase technique two weeks after the last inoculation. In addition, the mice were challenged by live influenza virus and the viral titer was measured 4 days postchallenge in the lungs of animals. The results of experiments demonstrated that intramuscular immunization of mice induces a stronger CTL response. Mice immunized by intramuscular route also showed a higher ability in virus clearance from the lungs. Results of this study showed that different routes of immunization of influenza NP genetic vaccine induce different levels of cell-mediated immune responses and protection from the live virus

[Analysis of antibody response to measles vaccine with censored data]

Analysis of measles serum antibody responses is an important tool for evaluating the level of immunity in vaccinated people and for determining the factors which influence protective immunity. Usually antibody concentration values which are determined by quantitative assays are left censored, so standard analysis such as simple linear regression model may not be appropriate. Measles antibodies were measured using Enzyme Linked Immunosorbent Assay [ELISA] in 200 person aged 5-25 years before and after vaccination. A censored regression model proposed in order to reduce the effects of censored data in parameter estimating. Twenty two persons [11 percent] of the sample had titer below detection limit which made censored data. 83 percent of children and teenagers [less than 16 years] and 88 percent of participant aged 16 and more were protective against measles disease, but there was no statistically significance between age and immunity [P= 0.4]. Although the number of females who had protective antibodies against measles was less than males, they possessed an average titer 42 percent higher than males [without considering covariates]. This proportion changed into 39% by using the censored regression model and adjusting with respect to [antibody titer before vaccination]. Both ratios showed higher vaccine- induced protection in females [P< 0.05]

Interferon resistance of hepatitis C virus genotypes 1a/1b: relationship to structural E2 gene quasispecies mutations

Hepatitis C virus [HCV] envelope glycoprotein-2 [E2] inhibits the interferon [IFN]induced, double stranded RNA activated protein kinase [PKR] via PKR eukaryotic initiation factor-2a phosphorylation homology domain [PePHD]. Present study examined the genetic variability of the PePHD in patients receiving interferon therapy. The PePHD region from HCV genotype 1a/1b infected patients receiving IFN was amplified by reverse transcriptase polymerase chain reaction [RT-PCR] and analyzed using bidirectionaly sequencing. The PePHD sequence was different in pretreatment isolates from three months treated patients. It was shown that the major PePHD quasispecies could change after three months IFN therapy and in one patient; the major PePHD quasispecies could change after six months IFN therapy. These mutations were occurred at codons 665, 666 and 667 of followed-up samples and at codons 660, 661, 666 and 670 of randomly treated patients. Some of these mutations were similar to those reported in previous studies. Other mutations were also detected in upstream and downstream regions of PePHD which may have influenced the structure, conformation and configuration of this region and thereby suppressing PePHD inhibitory properties. In conclusion our data suggested that HCV E2 PePHD may play an important role in determining the interferon response among Iranian HCV infected patients

use of a sensitive RT-Nested PCR method for detection of Hepatitis C virus

Hepatitis C virus is the major cause of viral hepatitis and its diagnosis in suspected specimens is of great importance. The risk of transfusion- transmitted virus infection is primarily the result of failure in serological screening tests to detect recently infected donors in the pre-seroconversion window period of infection. Therefore, sensitive and accurate diagnosis of HCV prior to antibody production to reduce window period is necessary. In the present study, a sensitive and specific RT-Nested PCR method for detection of a conserved HCV 5 UTR sequence was developed. Two pairs of primers for amplification of the target sequence in two rounds of PCR were selected. The developed RT- Nested PCR assay was performed on HCV-antibody confirmed positive samples as well as negative controls and standard samples. In order to compare the results, One Step RT-PCR kit was used in this study. 25 HCV-positive plasma samples whose positivity were confirmed by ELISA and Western Blot tests, also as well as 10 fold dilutions of a high viral load plasma sample obtained from a HCV-positive patient as standard samples and 25 negative control plasmas from healthy blood donors were collected and tested by this assay. In all of positive samples a 175bp band was observed on agarose gel electrophoresis, but no band could be detected in negative control plasma. Results from developed RT-PCR assay and One Step RT-PCR kit showed a good correlation. According to the results of this study, the developed RT-Nested PCR assay has a good sensitivity and specificity for diagnosis of HCV infection. It has the advantage of viral genome detection prior to seroconversion and can be used to detect HCV infection during window period

Application of SYBR-Green Real Time RT-PCR for quantification of human immunodeficiency virus type-1 [HIV-1] and comparison with COBAS AMPLICOR test

In this study, a SYBR Green real-time RT-PCR assay for quantification of HIV-1 viral RNA was developed. This assay was performed based on amplification of the pol region of HIV-1 and product analysis by an ABI 7500 system. We quantified HIV-1 viral load in 26_seropositive patients by this system and the data were subsequently compared with results obtained with a reference technique represented by COBAS AMPLICOR HIB-1 Monitor test. The results demonstrated that this technique could detect up to 500 HIV-1 RNA copies/ml of plasma. The linearity of this approach was conserved over a wide range of HIV-1 copy numbers [5x10[2]- 5x10[9]]. Since no positive signal was observed in seronegative volunteers, the specificity of the test was calculated as 100%. Comparison of the results with those obtained by the reference quantification method, revealed a significant correlation between the results [R[2] =0.95]. On the basis of the most recent recorded cases for HIV-1 infection and AIDS in Iran, the prevalence of this disease is rising rapidly and the situation has been called to be alarming by national health representatives. Determination of HIV-1 viral load in plasma has been considered as the most effective single prediction tool for monitoring HIV-1 patients treated with antiviral drugs. In this study, we have developed a SYBR-Green Real Time RT-PCR assay for quantitative analysis of HIV-1 in infected patients. Since a synthetic RNA standard was used in this assay, the upper limit of detection was detected to be higher than the standard test [5x10[9] versus 7.5x10[5]]. This can be important in patients with acute high viral load infections. Reproducibility was assessed by Intra assay and Inter assay analysis, Coefficient of variations Ct, in reproducibility tests for Intra assay and Inter assay variability were less than 3% and 4.5% accordingly. The above results, indicates that the new developed test can be a used in substitution of the commercial assay for quantitative analysis of HIV-1

Immune response analysis of immunized Balb/C mice with HIV-1 fusion p24 - gp41 genes as DNA vaccine candidate

The global HIV epidemic continues to expand and exceeding previous predictions. An effective vaccine represents the best hope to curtail the HIV epidemic. DNA vaccines induce humoral and cellular responses and mimic live vaccines without their pathogenic potential. The importance of CD8[+]CTL responses in controlling HIV and SIV viremia has led to production of a series of vaccines candidates that effectively induce these responses. It is now widely believed that an HIV vaccine strategy must stimulate both a strong humoral [antibody] as well as cell-mediated [CTL] immune response. The p24 and gp41 play many important roles in host-virus interaction and pathogenesis. These proteins are considered as attractive vaccine candidate in which their immunogenecity and immunomodulatory effects have been confirmed. In this study, a construct, pcDNA3.1Hygro- [p24-gp41], was evaluated as a DNA vaccine candidate in Balb/C mice for generation of effective cellular immune responses. For immunizing, we used dendrosome, a novel family of vehicles for transfection and therapy. IFN-gamma cytokine production and total antibody were detected by ELISA. Lymphoprolifration assay was performed by MTT test. ELISA and MTT assays confirmed that the cited p24-gp41 fusion gene is able to enhance immune responses in mice. The construct that was used in this research can be a good candidate for DNA vaccine against HIV-1, if the future complementary tests demonstrate the same trends of immunogenic responses shown in this study

Detection and genotyping of hepatitis D virus from HBsAg positive patients in Iran using RT-PCR

Hepatitis Delta virus [HDV] is a degenerate RNA virus or virusoid and a satellite of Hepatitis B virus [HBV]. Three distinct genotypes are described for HDV; genotype I is distributed worldwide but other genotypes appear to be more restricted geographically. In the present study, an RT-nested PCR method was set up to detect delta infection from serum samples. Moreover, the target amplified sequences corresponding to the Hepatitis delta antigen [HDAg] C-termini were used for genotyping. The results showed that 63.6% [23 of 36] of [HDAb] positive serum samples [as determined by ELISA] were also positive for HDV-RNA. Sequencing and phylogenic analysis of three Iranian HDV isolates revealed the most homology [93%] with an Italian isolate indicating a close relationship and probably a common origin for these isolates

Evaluation of a new dot blot assay for confirmation of human immunodeficiency virus type 1 and 2 infection using recombinant p24, gp41, gp 120 and gp36 antigens

Objectives:A sensitive and accurate dot blot assay using recombinant p24 [gag], gp41 and gp120 [env] proteins of HIV-1 and also recombinant gp36, the specific HIV-2 antigen was developed to confirm the presence of antibodies in sera reactive in screening enzyme-linked immunosorbent assays.Methods:We collected sera from Iranian 125 confirmed HIV positive Iranian samples [seropositive group] from AIDS patients, asymptomatic HIV-infected subjects, HIV-infected intravenous drug users and also hemophilic infected subjects. The samples were obtained from the AIDS Specimen Bank, Pasture Institute, Iran during 2002 to 2003. We also obtained 180 samples [seronegative group] from healthy blood donors. Recombinant antigens were expressed in Escherichia coli. By use of highly purified antigens, the dot blot procedure was developed. Analysis of the results was accomplished by capturing the dot blot images.Results:We established and interpreted the results using Centers for Disease Control criteria. We defined the positive test result as the presence of antibody against at least 2 different HIV gene products, one of which had to be an env gene product while a negative test result was defined as no antibodies against any of the HIV gene products and an indeterminate result was defined as antibodies reacting with only one HIV env gene product or against gag gene product only.Conclusion:The recombinant HIV dot blotting assay identified seropositive individuals with a high degree of accuracy; none of the HIV-seropositive subjects had a negative test result. Reactivity with these antigens, demonstrated 100% sensitivity and specificity in distinguishing seronegative from seropositive sera. The different sets of Western blot interpretative accepted criteria did not make a difference in interpretation of the seronegative and seropositive samples

Indeterminate human immunodeficiency virus western blot results in Iranian patients with discordant screening assay results

The Western blot [WB] assay is the most widely accepted confirmatory assay for the detection and confirmation of antibodies to human immunodeficiency virus type 1 [HIV-1] and 2 [HIV-2]. However, indeterminate WB reactivity to HIV-1 and HIV-2 proteins may occur in individuals who do not appear to be infected with HIV. In this study, we describe the results of indeterminate WB reactivity in Iranian patients with discordant screening assays. The samples were obtained from the Iranian Blood Transfusion Center, Tehran, Iran and evaluated in the Biotechnology Process Development Center, Pasteur Institute of Iran, Tehran, Iran between 2003 and 2004. A total of 4707 were tested for the presence of HIV-1 antibodies. Six hundred and four [12.8%] patients tested for HIV were positive for HIV-1 antibody. Nine [1.49%] have discordant results among screening assays and indeterminate WB results as interpreted by Centers for Disease Control and Prevention [CDC] criteria. Most [66.7%] of these indeterminate WB results were due to p24 reactivity. However, 2 [22.2%] display reactivity to both gp41 and gp120 proteins [Positive by World Health Organization [WHO] criteria]. Of 9 WB assays initially indeterminate by the CDC criteria and with follow-up samples, 8 [88.8%] became negative when retested subsequently while one [11.1%] remained indeterminate for more than a year and were thus considered negative. In addition, all the indeterminate samples were negative when assessed by polymerase chain reaction assay. In general, there was an 88.8% concordance between the CDC and WHO criteria for an indeterminate WB result. The CDC II criteria best met the specified objectives for diagnosis in our setting