First Detection of Human Polyomaviruses in HIV Patients with Suspected Neurological Disease in Montería, Colombia

Objective: The objective is to establish the presence of JC and/or BK polyomavirus in HIV patients with symptoms of encephalitis and/or meningitis. Methodology: From September 2009 to December 2011, a prospective study was conducted. 34 HIV patients with symptoms consistent with encephalitis and/or meningitis were included. The work was conducted in 3 hospitals in the city of Monteria. Viral DNA extraction was performed on samples of cerebrospinal fluid (CSF) using a commercial kit (Quiagen, USA). The detection of BKV and JCV was performed by multiplex real-time PCR (LightMix ?, Roche Diagnostics, Germany) with primers specific for the short t antigen gene fragment, labeled probes and one internal control. Results: In 9 (26%) of 34 patients included in the study, JCV virus was detected; only 1 (3%) patient had coinfection with JCV/BKV. The mortality rate was 3%. The cytochemical examination of CSF in positive patients presented average values: 40.7 mg/dL glucose, 171.66 mg/dL protein, 19.8 mm ³ leukocytes, and 109.8 mm ³ erythrocytes. Conclusion: The findings demonstrate that JCV and BKV have a significant occurrence in HIV patients with CSF infections in Monteria.

• Progressive Multifocal Leukoencephalopathy
• Meningitis
• Encephalopathy
• AIDS

1. Introduction

Human polyomaviruses belong to the family Papovaviridae. BK virus (BKV) and JCV virus are the principal members of this group that affect human beings [1] [2]

Nearly 70% to 80% of healthy people have antibodies against BKV and JCV [3] [4] . Initial infection with both BKV and JCV occurs commonly in childhood or in adolescence and is generally asymptomatic [5] . These viruses can persist latently in many sites, including kidneys, CNS and lymphoid cells. Recrudesce of latent infection may then occur in immunocompromised individuals [6] .

The occurrence of BKV and JCV in people infected with human immunodeficiency virus (HIV) ranges from 20% - 44% [7] . In immunecompromised patients, JCV is the main etiologic agent of progressive multifocal leukoencephalopathy (PML), a fatal neurological disease, as well as granular cell neuropathy, encephalopathy and meningitis, the BKV is associated with hemorrhagic cystitis in stem cell transplantation (HSCT) hematopoietic and interstitial nephropathy in kidney transplant recipients, it has recently been shown that is associated with neurological complications [4] [8] [9] .

Nonetheless, PML prevalence frequently increases during AIDS epidemics, where up to 5% of AIDS patients may develop the disease. In fact, the mortality associated with PML has also expanded from 1.5 per ten million people in the initial HIV era to 6.1 deaths per ten million persons in the post HIV era [9] .

Currently, a new PML presentation has appeared among patients managed with immunomodulatory drugs for autoimmune diseases [9] - [11] . BKV is associated with hemorrhagic cystitis in hematopoietic stem cell transplantation recipients and interstitial nephropathy in kidney transplant recipients and has lately also been demonstrated to be related to neurologic exacerbations [4] .

There are about 1.7 million people infected with HIV in Latin America and 250,000 in the Caribbean in Colombia in 2014, In Cordoba, 9700 cases were reported, with a mortality rate of 6.7% and 380 of these cases were notified as HIV/AIDS positive [12] - [14] .

Between 1992-2000 in Brazil and Mexico, the prevalence of PML in postmortem samples of patients confirmed with AIDS was between 0.8% and 3% [15] . In Colombia, only four cases of polyomavirus infection have been reported, Triana et al., [16] reported the first case of PML in Latin America and Colombia (Bogotá) associated with natalizumab in a patient with a diagnosis of multiple sclerosis; the virus was identified in brain biopsy using the technique of in situ hybridization. In Cali, Cadavid et al. [17] studied two patients with kidney transplants; BKV was identified by immunohistochemistry in a kidney biopsy. However, there are no studies in Colombia on polyomavirus in HIV individuals associated with neurologic disease.

The objective of this study was to detect JCV and BKV in HIV patients with symptoms consistent with encephalitis and/or meningitis.

2. Materials and Methods

2.1. Patients and Specimens

34 CSF specimens were collected from HIV patients with suspected meningitis and/or encephalitis between September 2009 and December 2011, at the San Jerónimo of Monteria hospital main clinical center of third level of care of the department of Cordoba. 25 patients were males and 9 females aged 3 - 63 years. CSFs were frozen at −80˚C until processing. The selection of patients was independent of group age, gender and membership in health system, the selection criteria were: patients with diagnosis of suspected encephalitis [18] was defined as: fever, headache, convulsions, signs of brain involvement, such as alteration of consciousness and/or personality, seizures and focal neurologic signs and the diagnosis of suspected meningitis [19] was defined as: fever, headache, neck stiffness.

2.2. Molecular Detection of JC and BK Viruses

Polyomavirus DNA was extracted from 200 μl of CSF samples using a resin column kit (Quiagen, USA Cat. 51306) according to the manufacturer’s instructions. The DNA was kept at −20˚C until processing. To detect JCV and BKV a kit LightMix® polyomaviruses JC and BK (Cat-40-0203-32, Roche Diagnostics, Tibmolbiol), multiplex PCR in real-time was performed. The kit amplifies a fragment of the gene encoding t antigen 175 bp (BKV) and 172 bp (JCV). In each test negative and positive controls and internal standards were included; those were used to generate viral load curves for JCV and BKV (10¹ to 10⁶).

2.3. Laboratory Data

The CSFs were analyzed by: tests of glucose and protein levels, cell count of leukocytes and erythrocytes, Chinese ink, Gram staining, Ziehl-Neelsen and culture.

2.4. Ethics

Institutional standard guidelines of the Minister of Health of Colombia and University of Cordoba ethics committee were followed for the collection of patient’s CSF samples after obtaining their written informed consent for involvement in the study. All ethics issues were according to the resolution number 8430 of 1993 [20] . During the study confidentiality of the information was maintained.

2.5. Statistics

Chi-square test and ANOVA were used for statistical analysis. Analysis was performed in EpiInfo Version 7.1 (CDC, Atlanta) software. P < 0.05 was considered significant.

3. Results

Between September 2009 and December 2011, 34 HIV patients with neurological manifestations were studied. The median age of the cohort was 33.5 years (range, 3 - 63 years); of these 34 patients 25 (74%) were male and 9 (26%) were female.

3.1. Clinical Features

The following clinical manifestations were found in 34 patients: 28 (82%) patients presented with headache, 20 (59%) fever, 14 (41%) nausea, 12 (35%) neck rigidity, 11 (33%) convulsions, 8 (24%) altered mental status, 7 (21%) decrease of the muscular force, 4 (12%) irritability, 3 (9%) disartria, 2 (6%) hipoestesia and 1 (3%). flaccid paralysis. CSF samples showed opportunistic pathogens in 6 of 34 (18%) patients; 4 (12%) patients had Cryptococcus neoformans and 2 (6%) were infected by Treponema pallidum. The hematological and biochemical parameters were as follows: leukocyte count (average 54.03/mm³), predominantly mononuclear (average 15%), erythrocyte count (average 44.96 mm³), glucose levels (average 35.12 mg/dl), and increase in protein (average 130.11 mg/dl).

3.2. Detection of JCV and BKV in CSF

JCV was detected in 9/34 patients (26%), with a mean viral load of 1.535 copies/µl (range, 500 ? 1,000,000 copies/µl). One patient (3%) had coinfection with JCV/BKV; a viral load of 850 copies/µl of JCV and 650 copies/µl of BKV were found. Viral load, PCR and hematological and biochemical parameters are described in Table 1. There were no significant differences in the age, sex, clinical manifestations and patients with infection by JCV and BKV.

3.3. Clinical Presentation of Patients with Infection by JCV and BKV

The median age was 35 years (range, 3 - 62 years). Only one of 34 (3%) patients died with infection by JCV (Table 1). Patients with JCV or BKV infection presented the following signs and symptoms: headache (80%), fever (70%), seizures (40%), vomiting (40%) and nausea, decreased muscle strength, nuchal rigidity and hypoesthesia, altered mental status were seen with less frequency (Table 1). Ten patients showed glucose average of 40.7 mg/dl, protein levels had an average of 171.66 mg/dl, protein levels were elevated in only 3 patients. Regarding hematological patterns, the leukocytes had an average of 19.8 mm³, erythrocytes had an average of 109.8 mm³, predominantly mononuclear 10%, in 3 patients the leukocyte count was elevated (Table 1).

4. Discussion

This is the first prospective study carried out in Colombia in HIV patients infected by JCV and BKV; all HIV patients enrolled in the study had suspected meningitis or encephalitis. The overall prevalence was 26% for polyomavirus and 3% of coinfection with JCV/BKV. The frequency of infection with JCV was significantly higher than other studies, which reported infection rates of 6% with JCV and coinfections with JCV/BKV in only 3.3% (Table 1). In several studies carried out in urine samples, cervical carcinoma, semen and saliva, the detection

References

1. Pinto, M. and Dobson, S. (2014) BK and JC Virus: A Review. Journal of Infectio, 68, S2-S8.

2. Dekeyser, M., Francois, H., Beaudreuil, S. and Durrbach, A. (2015) Polyomavirus-Specific Cellular Immunity: From BK-Virus-Specific Cellular Immunity to BK-Virus-Associated Nephropathy? Frontiers in Immunology, 6, 307. http://dx.doi.org/10.3389/fimmu.2015.00307

3. Jiang, M., Abend, J.R., Johnson, S.F. and Imperiale, M.J. (2009) The Role of Polyomaviruses in Human Disease. Virology, 384, 266-273. http://dx.doi.org/10.1016/j.virol.2008.09.027

4. Nali, L.H., Centrone Cde, C., Urbano, P.R., et al. (2012) High Prevalence of the Simultaneous Excretion of Polyomaviruses JC and BK in the Urine of HIV-Infected Patients without Neurological Symptoms in Sao Paulo, Brazil. Revista do Instituto de Medicina Tropical de Sao Paulo, 54, 201-205. http://dx.doi.org/10.1590/S0036-46652012000400004

5. De Gascun, C.F. and Carr, M.J. (2013) Human Polyomavirus Reactivation: Disease Pathogenesis and Treatment Approaches. Clinical & Developmental Immunology, 2013, Article ID: 373579. http://dx.doi.org/10.1155/2013/373579

6. Behzad-Behbahani, A., Klapper, P.E., Vallely, P.J., Cleator, G.M. and Bonington, A. (2003) BKV-DNA and JCV-DNA in CSF of Patients with Suspected Meningitis or Encephalitis. Infection, 31, 374-378.

7. Machado, D.M., Fink, M.C., Pannuti, C.S., et al. (2011) Human Polyomaviruses JC and BK in the Urine of Brazilian Children and Adolescents Vertically Infected by HIV. Memórias do Instituto Oswaldo Cruz, 106, 931-935. http://dx.doi.org/10.1590/S0074-02762011000800006

8. Verbeeck, J., Van Assche, G., Ryding, J., et al. (2008) JC Viral Loads in Patients with Crohn’s Disease Treated with Immunosuppression: Can We Screen for Elevated Risk of Progressive Multifocal Leukoencephalopathy? Gut, 57, 1393-1397. http://dx.doi.org/10.1136/gut.2007.145698

9. Tan, C.S. and Koralnik, I.J. (2010) Progressive Multifocal Leukoencephalopathy and Other Disorders Caused by JC Virus: Clinical Features and Pathogenesis. The Lancet Neurology, 9, 425-437. http://dx.doi.org/10.1016/S1474-4422(10)70040-5

10. Boothpur, R. and Brennan, D.C. (2010) Human Polyomaviruses and Disease with Emphasis on Clinical BK and JC. Journal of Clinical Virology, 47, 306-312. http://dx.doi.org/10.1016/j.jcv.2009.12.006

11. Iannetta, M., Bellizzi, A., Lo Menzo, S., et al. (2013) HIV-Associated Progressive Multifocal Leukoencephalopathy: Longitudinal Study of JC Virus Non-Coding Control Region Rearrangements and Host Immunity. Journal of NeuroVirology, 19, 274-279. http://dx.doi.org/10.1007/s13365-013-0167-9

12. Instituto Nacional de Salud. Informe del comportamiento en la notificación de vih-sida hasta el periodo epidemiológico 13 del ano 2014. Informe de Evento, FOR-R02.4000-001(2), 1-22. http://www.ins.gov.co/lineas-de-accion/Subdireccion-Vigilancia/Informe%20de%20Evento%20Epidemiolgico/VIH%20SIDA%20%202014.pdf

13. Instituto Nacional de Salud, Dirección de Vigilancia y Analisis Del Riesgo en Salud Pública, Sistema de Vigilancia en Salud Pública (SIVIGILA). Boletin epidemiologico semanal, Semana epidemiológica número 53 de 2014 (28 dic. al 03 ene 2015). http://www.ins.gov.co/boletin-epidemiologico/Boletn%20Epidemiolgico/2014%20Boletin%20epidemiologico%20semana%2053.pdf

14. Instituto Nacional de Salud, Dirección de Vigilancia y Analisis Del Riesgo en Salud Pública, Sistema de Vigilancia en Salud Pública (SIVIGILA). Estadísticas. Vigilancia rutinaria. Casos confirmados VIH/SIDA por departamento ano 2014. http://www.ins.gov.co/lineas-de-accion/Subdireccion-Vigilancia/sivigila/Paginas/vigilancia-rutinaria.aspx

15. Khalili, K. and Stoner, G.L. (2004) Human Polyomaviruses: Molecular and Clinical Perspectives. John Wiley & Sons, New York.

16. Triana, J., Reyes, M., Hernández, L., Mendoza, O., Salgado, S. and Becerra, G.P. (2014) Leucoencefalopatía multifocal progresiva asociada al uso de natalizumab en un paciente con esclerosis múltiple. Primer caso en Latinoamérica. Hospital de San José—Bogotá 2013. Acta Neurológica Colombiana, 30, 200-204.

17. Cadavid, D., Mesa, L., Schweineberg, J. and Posada, J.G. (2015) Polyomavirus-Associated Nephropathy in Renal and Simultaneous Pancreas-Kidney Transplant Recipients: Report of Three Cases. Saudi Journal of Kidney Diseases and Transplantation, 26, 94-97. http://dx.doi.org/10.4103/1319-2442.148750

18. Venkatesan, A., Tunkel, A.R., Bloch, K.C., et al. (2013) Case Definitions, Diagnostic Algorithms, and Priorities in Encephalitis: Consensus Statement of the International Encephalitis Consortium. Clinical Infectious Diseases, 57, 1114-1128. http://dx.doi.org/10.1093/cid/cit458

19. Swanson II, P.A. and McGavern, D.B. (2015) Viral Diseases of the Central Nervous System. Current Opinion in Virology, 11, 44-54. http://dx.doi.org/10.1016/j.coviro.2014.12.009

20. Republica de Colombia, Ministerio de Salud. Resolución No. 008430 del 4 de octubre del 1993. Por la cual se establecen las normas científicas, técnicas y administrativas para la investigación en salud, 1993.

21. Wiedinger, K., Bitsaktsis, C. and Chang, S. (2014) Reactivation of Human Polyomaviruses in Immunocompromised States. Journal of NeuroVirology, 20, 1-8. http://dx.doi.org/10.1007/s13365-014-0234-x

22. Karalic, D., Lazarevic, I., Knezevic, A., Cupic, M., Jevtovic, D. and Jovanovic, T. (2014) Distribution of JC Virus Genotypes among Serbian Patients Infected with HIV and in Healthy Donors. Journal of Medical Virology, 86, 411-418. http://dx.doi.org/10.1002/jmv.23796

23. Netravathi, M., Mahadevan, A., Satishchandra, P., et al. (2013) Progressive Multifocal Leukoencephalopathy (PML) Associated with HIV Clade C—Is Not Uncommon. Journal of NeuroVirology, 19, 198-208. http://dx.doi.org/10.1007/s13365-013-0168-8

24. Berger, J.R. (2014) Progressive Multifocal Leukoencephalopathy. Handbook of Clinical Neurology, 123, 357-376. http://dx.doi.org/10.1016/B978-0-444-53488-0.00017-1

25. Palazzo, E. and Yahia, S.A. (2012) Progressive Multifocal Leukoencephalopathy in Autoimmune Diseases. Joint Bone Spine, 79, 351-355. http://dx.doi.org/10.1016/j.jbspin.2011.11.002

26. Wang, Y., Kirby, J.E. and Qian, Q. (2009) Effective Use of JC Virus PCR for Diagnosis of Progressive Multifocal Leukoencephalopathy. Journal of Medical Microbiology, 58, 253-255. http://dx.doi.org/10.1099/jmm.0.004432-0

27. Robaina, T.F., Mendes, G.S., Benati, F.J., et al. (2013) Polyomavirus in Saliva of HIV-Infected Children, Brazil. Emerging Infectious Diseases, 19, 155-157. http://dx.doi.org/10.3201/eid1901.120563

28. Comar, M., Zanotta, N., Croci, E., et al. (2012) Association between the JC Polyomavirus Infection and Male Infertility. PLoS ONE, 7, e42880. http://dx.doi.org/10.1371/journal.pone.0042880

29. Behzad-Behbahani, A., Klapper, P.E., Vallely, P.J., Cleator, G.M. and Bonington, A. (2003) BKV-DNA and JCV-DNA in CSF of Patients with Suspected Meningitis or Encephalitis. Infection, 31, 374-378.

30. Siddiqi, O.K., Ghebremichael, M., Dang, X., et al. (2014) Molecular Diagnosis of Central Nervous System Opportunistic Infections in HIV-Infected Zambian Adults. Clinical Infectious Diseases, 58, 1771-1777. http://dx.doi.org/10.1093/cid/ciu191