Rajesh Kannangai*, Jaiprasath Sachithanandham*, Anita Mahadevan**, Asha Mary Abraham*, Gopalan

Transcription

1 JCM Accepts, published online ahead of print on 2 January 2013 J. Clin. Microbiol. doi: /jcm Copyright 2013, American Society for Microbiology. All Rights Reserved Association of neurotropic viruses in HIV infected Individuals died of secondary complications of tuberculosis, cryptococcosis or toxoplasmosis: A study from India (South) Rajesh Kannangai*, Jaiprasath Sachithanandham*, Anita Mahadevan**, Asha Mary Abraham*, Gopalan Sridharan #, Anita Desai ##, Vasanthapuram Ravi ##, Susarla Krishna Shankar** * Department of Clinical Virology, Christian Medical College, Vellore ** Department of Neuropathology, NIMHANS, Bangalore # Sri Sakthiamma Biomedical Research Institute, SNHRC, Vellore ǂ Department of Neurovirology, NIMHANS, Bangalore Corresponding Author Rajesh Kannangai, MD, PhD Professor, Department of Clinical Virology Christian Medical College, Vellore , India ID: kannangair@cmcvellore.ac.in Phone # Fax #

2 Abstract: The frequency of 10 opportunistic DNA viruses was determined by multiplex real-time PCR in paired CSF and brain tissue of HIV infected individuals. In the CSF viruses were detectable in 45/55 cases, JCV (62%), EBV (44%) and CMV (25%), followed by VZV (3.6%), HSV -1 (1.8%) and HHV-6 (1.8%). Single virus was detectable in 20, 19 had co-infection with two viruses, 6 were positive for three viruses. JCV was detectable in CSF of 62% and 42% of brain tissues with higher loads in PML (p value <0.05). Downloaded from on November 18, 2018 by guest 2

3 Several viruses can cause opportunistic infections in HIV infected individuals with high morbidity and mortality in HIV infected individuals (1). These infections are more frequently reactivation of the latent viruses in the host. It is essential to identify the causative agent of these opportunistic infections to initiate therapy or prophylactic treatment. As the clinical picture caused by different viruses often overlap and multiple infections are also frequent in immunocompromised host, simultaneous screening of a wide range of viruses would be cost effective management (2). Viruses like Epstein-Barr virus (EBV), cytomegalovirus (CMV), and JC virus (JCV) can result in life-threatening infections in the central nervous system (CNS) of immunodeficient individuals (3, 4). The clinical conditions include primary CNS lymphoma, cytomegalovirus encephalitis, and progressive multifocal leukoencephalopathy (PML) (5-7). To compound the clinical problem, viral infections can coexist with bacterial and fungal infections. Currently there are no reports from India on the frequency of CNS viral infections among HIV -1 infected individuals. The aim of this study was to develop a real-time multiplex PCR for the simultaneous detection of DNA viruses in paired CSF and brain tissue samples of HIV infected individuals. The study was carried out in the Departments of Clinical Virology (site 1), and Neuropathology (site 2), of two tertiary care centers in south India. The study was approved by the institutional review board of both the institutions. Brain tissues and CSF samples of histopathologically well characterized HIV associated opportunistic infections collected at autopsy and archived in Human Brain Tissue Repository of Neuropathology was utilized for the study. All the samples archived are collected at autopsy following written informed consent from the legal close relative(s) of the deceased to use the biological material for research. A total of 55 archived paired CSF and brain tissue samples collected from HIV positive individuals who had died with confirmed bacterial or fungal infections were investigated. These diagnoses were confirmed by histopathology, immunohistochemistry and or microbiological methods (culture, serology). The study also included 20 CSF samples collected from patients who died of noninfectious disease like malignancy or road 3

4 55 56 traffic accidents. Brian tissue samples were available from 10 of these individuals. These samples were used as disease free controls DNA were extracted from CSF sample using the commercially available QIA amp DNA blood mini kit (Qiagen, Hilden, Germany) and from brain tissue samples using QIA amp DNA mini kit (Qiagen, Hilden, Germany). Sterile Milli-Q water used as a negative control and was included after every 5 th sample in each run of extraction to check the PCR cross contamination. The Multiplex real time was done on PCR Rotor gene RG- 3000/6000 (Corbett Research, Sydney Australia). The primers were used in four cocktail mixes as shown: Cocktail-1: CMV (8), EBV (8) and HSV-1(8), Cocktail-2: VZV (8) and HHV-6 (8), Cocktail-3: JC (8) and HSV-2 (8). Cocktail-4: HHV-8 (8), BK (9) and Adenovirus (10). The samples found positive for respective viruses in initial screening multiplex PCR were tested by individual in-house quantitation assays. DNA integrity of the all PCR negative samples was confirmed by testing for a 135bp segment of the human endogenous retrovirus 3 (ERV-3) envelope gene (11) and the ERV-3 assay is also used to determine the copy numbers of viruses in tissue. The lower detection limits for all the viruses used in the study were as follows; HSV TCID 50 /10µl input (0.56TCID 50 /ml), HSV TCID 50 /10µl input (0.31TCID 50 /ml), VZV PFU/10µl input (0.1 PFU/ml), EBV - 1 plasmids/ 10µl input (<100 copies/ml), CMV - 1 to 10 copies/10µl input ( copies/ml), HHV-6-1 plasmids /10µl input (<100 copies/ml), HHV-8-45 plasmids/ 10µl input (4500 copies/ml), JCV - 50 plasmids/10µl input (5000 copies/ml) and BKV - 1 plasmids/ 10µl input (<100 copies/ml). 75 Genotyping was carried out for JCV, EBV and CMV by amplifying 610 bp IG region for JCV, for CMV bp gb region and for EBV amplifying a 447 bp of LMP-2A gene (12-14). The sequencing of these amplified products was done in an ABI 310 automated sequencer (Applied Biosystems, Foster City, CA). The 4

5 78 79 phylogenetic tree analyses was carried out by the neighbor joining method using 1000 bootstrap replicates in MEGA4 software All the PCR negative samples amplified for ERV-3 real time PCR confirming sample DNA integrity. In the 55 paired CSF and tissue samples tested, three of the most common viruses detected were JCV, EBV and CMV whereas VZV, HSV-1 and HHV-6 were less common. Comparison of positivity rate of viruses in paired samples showed higher frequency in CSF than brain tissues [CSF versus tissue positivity: JCV- 62% vs 42%, CMV- 25% vs 18%, EBV- 44% vs 31%). Overall, in CSF viruses were detected 45/55 samples tested [monomicrobial in 20 and poly-microbial combination of two or three viruses in 25]. Of the mono-microbial agents, JCV was most frequent (10/20), followed by EBV and CMV (5 each). In the polymicrobial group (n=25), JCV was again the most prevalent virus [24/25]. In nineteen individuals, coinfection with two viruses was seen in the CSF. In eighteen of these, JCV was positive, in combination with EBV (n 14 cases), CMV (in 3 cases) and HSV-1 (in 1 case). The remaining one case showed co-existence of EBV with HHV-6. In six individuals, co-infection with three viruses was detected. JCV was found in all six cases. In four, JCV co-infection was seen with EBV and CMV, while in the remaining two, JCV was seen in combination with CMV and VZV. In tissue samples, viruses were detected in 37/55 samples tested [mono-microbial in 19, poly-microbial in 18]. In 19 cases wherein single virus was detected, JCV/EBV were seen in 7 cases each, CMV in four and HHV-6 in one case. Of 18 individuals with poly-microbial infection, 17 had dual infection with two viruses and in one individual co-infection with three viruses were seen. Of the 17 cases with dual infection, JCV was most frequently detected (15 of 17), co-infected with EBV in 8, CMV in 4 and HSV-1/VZV or HHV-6 in one case each. In the remaining two cases, combination of EBV with CMV or EBV with HHV-6 was detected. The single individual with triple infection had combination of JCV with EBV and HHV-6. 5

6 None of the samples were positive for HHV-8, HSV-2, BK virus and adenovirus. Among the 20 controls tested, a single CSF sample was positive for HHV-6 with viral load of copies/ml The EBV mean viral load in both CSF and tissue was very low (2.94 in CSF and 1.03 in tissue) compared to other viruses like JCV, CMV, VZV, HSV-1 and HHV-6. The data on the positivity rate for each virus in tissue and CSF with their mean viral load is shown in Table 1. The highest mean viral load in both CSF and brain was highest with JCV, followed by EBV and CMV. The PCR positive rate and the mean viral load for EBV, CMV and JCV in paired CSF and tissue samples are shown in Table 2. When the JCV, EBV and CMV mean viral load levels in CSF (copies/ml) were compared with respective mean virus load in brain tissue (copies/10000 cells) the viral load levels were significantly (p = <0.001) high in the CSF for all three viruses and this data is shown in Fig. 1. When the viral load levels in paired CSF and brain tissue samples was analyzed with Spearman s rank (rho) test there was a significant positive correlation in levels of JCV ( r= , p = 0.01). JCV viral loads in both CSF (mean viral load 7.96 log) and tissue (mean viral load 6.06 log) samples were significantly high in confirmed cases of PML (p value range from ). In other opportunistic infections, the EBV virus load was found to be significantly high (p = 0.031) in CSF of cases of tuberculosis, compared to PML. The percentage positive and the mean viral load level of JCV in both CSF and tissue samples were significantly higher (p < 0.002) than CMV and EBV. The EBV and CMV in-house quantitation assays were also validated with 6 samples for EBV and 4 samples for CMV received through UKNEQAS program. DNA from 24 JC positive, 14 EBV positive and 9 CMV positive samples were used for genotyping. All the 24 JCV strains were found to be genotype 2D. Out of 14 EBV strains 8 were found to be EBV genotype 2 and 6 were EBV genotype 1. Out of 9 CMV strains 8 were found to be genotype gb1 and 1 was found to be genotype gb2. 6

7 One of the important findings in our study is that viruses like EBV, CMV and JC virus were commonly found in the HIV infected individuals who succumbed to various bacterial, fungal or parasitic infections. In an earlier study from India, 147 CSF samples from various neurological conditions in non HIV infected individuals using real time multiplex PCR had reported the presence of multiple viruses in a single sample with EBV(34%) being most frequent (15). A study from Italy of herpes virus DNA in CSF of AIDS patients with CNS disease found one or more of the following viruses in 26 of 49 patients (53%); CMV in 16 (33%), EBV in 13 (27%), HHV-6 in 2 (4%), HHV-7 in 1 (2%), and HHV-8 in 1 (2%). CMV was associated with occurrence of encephalitis and peripheral neuropathy and EBV with primary CNS lymphoma (16). The study also reported 7 (14%) patients had DNA for more than one herpes virus (16). In another study from Italy employing nested PCR on CSF in HIV infected individuals, reported co-infections with more than one virus in CSF in 181 out of 500 patients (17). In our study only one (5%) of the 20 CSF of the control group was positive for HHV-6. These results show that the assays we had used in this study are very robust and specific. The higher proportion of positivity in our study population may be due to sampling in the late stage of AIDS. Significantly a high viral load of JCV in PML suggests that a critical threshold may be required for clinical manifestation with disease. The detection of multiple viruses in a given sample reflects the biological phenomena of transactivation which needs to be explored further. In our study we also examined the genotypes of the three common viruses seen in our population. A study that reported on the molecular characterization of JC virus in Brazilian HIV infected individuals with and without PML, reported JC virus genotypes 2 (32.7%), 1 (26.5%) and 3 (23.5%) in their population from CSF and urine samples. They also reported that JCV genotype 1 showed a strong association with PML (p<0.0001) while JCV genotype 3 had an inverse association with PML (18). Agostini et al (1998) had found JC virus genotype 2B to be more frequent (3.6%) in brain tissues of patients with PML compared to urine from controls (5.9%) and genotype 2B predicts the highest risk of PML disease in HIV infected individuals (19). In our study population 7

8 we observed only a single type of JC genotype i.e 2D in CSF and brain tissues samples of both cases with and without PML Several studies on EBV genotypes in EBV related diseases like Hodgkin's disease, Burkitt s lymphoma, Hodgkin's lymphoma and oral hairy leukoplakia had reported a higher percentage EBV genotype-1 with 56% (20), 58% (21), 82% (22) and 93% (23) respectively and in the same clinical conditions the EBV genotype-2 frequencies were 7% (23), 9% (22), 13% (20) and 23% (21) respectively. In contrast to these studies our study population had more or less same frequency of EBV genotype 2 (57%) and EBV genotype 1 (43%). A PCR based RFLP study from India (Chennai) in patients with CMV disease reported CMV gb2 and gb3 in 53.8 and 46.1 % of the patient s studied respectively (24). Our data showed a high frequency of CMV genotype gb1 (89%) followed by gb2 genotype (11%). This study emphasises the utility of multiplex real-time PCR assays in the simultaneous detection of multiple viral agents. The presence of multiple viruses in the same sample suggests one infectious agent leading to the reactivation of several other latent viruses and is of clinical relevance, essential for determining patient management. Acknowledgements We gratefully acknowledge the funding received from Department of Biotechnology (Govt. of India), for the study. The authors also thank Human Brain Tissue Repository (HBTR or Brain Bank), National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore for providing the CSF and brain tissue samples for the study. Data presented in this manuscript forms part of the PhD thesis of Mr. Jaiprasath Sachithanandham. The authors report they have no conflict of interest

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13 Table 1: Percentage positive and the mean viral loads detected in the paired CSF and tissue samples Paired samples (n=55) CSF Virus POS (%) Viral load* POS Tissue Viral load* $ Mean ± SD (%) Mean ± SD JCV 34 (62) 5.50± (42) 3.19 ± 2.48 EBV 24(44) 2.94± (31) 1.03± 0.70 CMV 14 (25) 3.16± (18) 1.17 ± 0.92 VZV 2(3.6) 7.00±2.31 1(1.8) 5.61 HSV-1 1(1.8) (1.8) 3.48 HHV-6 1(1.8) (7.3) 2.72± 0.17 None of the samples were positive for HHV-8, HSV-2, BK virus or adenovirus. * log 10 values. $ The viral load expressed as log copies/10000 cells based on ERV-3 copies.

14 Table 2: Total number of paired samples which were positive for viruses from various opportunistic infections: CSF n=55 Tissue* n=55 CM n=15 TE n=17 TBM n=11 PML n=7 CM n=15 TE n=17 TBM n=11 PML n=7 EBV POS (%) 6 (40) 7(41) 7 (64) 2(28) 6(40) 4(23) 4(36) 1 (14) Viral load Mean ± SD CMV POS (%) 2(13) 7(41) 1(9) 3(43) 3(20) 1(5.9) 1(9) 2(28) Viral load Mean ± SD JCV POS (%) 10(67) 7(41) 8 (73) 7(100) 5(33) 4(23) 5(45) 7(100) Viral load Mean ± SD All viral load values expressed as log 10 ; *The viral load expressed as log copies/10000 cells based on ERV-3 copies [The inter and intra-assay variations of the ERV-3 gene assay was estimated and was found to be < 0.3 log copies/ml]. CM- Cryptococcal meningitis, TE- Toxoplasma encephalitis, TBM- Tuberculous Meningitis, PML-Progressive multifocal leucoencephalopathy

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16 Fig 1 Comparison of EBV, CMV and JCV virus loads in paired CSF and tissue samples among HIV-1 infected individuals. (a) EBV (b) CMV (c) JCV a) b) EBV CSF EBV Tissue 0 CMV CSF CMV Tissue Two tailed t test probability- p= <0.001 Two tailed t test probability- p= <0.001 c) JC CSF JC Tissue Two tailed t test probability- p= <