Molecular epidemiology and risk factors associated with BK and JC polyomavirus urinary shedding after kidney allograft.

Polyomaviruses BK (BKPyV) and JC (JCPyV), belonging to the Polyomaviridae, are responsible for human pathologies. In kidney transplant recipients, BKPyV replication can lead to irreversible nephron damage whereas JCPyV replication remains asymptomatic. Concomitant replication is rare and potential competition between the infections has been described. The aim of this retrospective case-control study was to describe the molecular epidemiology and risk factors associated with BKPyV and JCPyV replication in a cohort of kidney transplant recipients. In total, 655 urine samples from 460 patients were tested for BKPyV and JCPyV DNA. Positive samples were submitted to strain genotyping. Demographic and clinical characteristics were also compared. Isolated JCPyV and BKPyV was found in 16.5% and 23.3% of patients, respectively; co-replication was rare (3.9%). BKPyV strains Ib-2, Ib-1, and IVc-2 were the most prevalent. JCPyV strains mostly belonged to genotypes 4 and 1B. During follow-up, JCPyV shedding significantly reduced the risk of BKPyV DNAuria, with an odds ratio of 0.57 (95% confidence interval: 0.35-0.99), and was associated with better prognosis than BKPyV replication, based on the estimated glomerular filtration rate. Molecular epidemiology of BKPyV and JCPyV strains in our region was similar to previous studies. This study suggests that JCPyV is benign and appears to limit damaging BKPyV replication. JCPyV DNAuria screening could thus be a useful strategy to predict BKPyV-related outcomes.

Testing for Human Papillomaviruses in Urine, Blood, and Oral Specimens: an Update for the Laboratory.

Twelve high-risk alpha human papillomavirus (HPV) genotypes cause approximately 690,000 cancer cases annually, with cervical and oropharyngeal cancer being the two most prominent types. HPV testing is performed in laboratory settings for various applications of a clinical, epidemiological, and research nature using a range of clinical specimens collected by clinicians or by individuals (self-collected specimens). Here, we reflect on the importance and justification of using the right test for the right application and provide practical updates for laboratories either participating in or anticipating involvement in HPV testing in three specimen types, namely, urine, blood, and oral specimens, which are considered "alternative" specimens by many. In addition to clinician-collected cervical samples and self-collected cervicovaginal samples, first-void urine is emerging as a credible specimen for HPV-based cervical cancer screening, triage of HPV screen-positive women, monitoring HPV vaccine impact, and HPV testing in groups for which a less invasive sample is preferred. Detection of cell-free DNA (including HPV DNA) in blood has great promise for the early detection of HPV-attributable oropharyngeal cancer (HPV-AOC) and potentially other HPV-driven cancers and as an adjunct prognostic marker in long-term tumor surveillance, including treatment response. The moderate sensitivity of HPV testing in oral rinses or swabs at HPV-AOC diagnosis prevents its use in HPV-AOC secondary prevention but represents a promising prognostic tool in HPV-AOC tertiary prevention, where the HPV persistence in oral rinses throughout treatment may predict early HPV-AOC recurrences and/or the development of secondary HPV-AOC. The increasing sophistication of specific collection devices designed for alternative samples and the enhanced precision of novel molecular technologies are likely to support the evolution of this field and catalyze potential translation into routine practice.

Quantitative assessment of viral load in the blood and urine of patients with congenital cytomegalovirus infection using droplet digital PCR.

Congenital cytomegalovirus infection (cCMV) is a common cause of congenital infections, leading to neurodevelopmental sequelae. Real-time quantitative polymerase chain reaction (qPCR) has been widely used for the diagnosis and assessment of cCMV; however, the correlation between CMV DNA load and the severity of cCMV symptoms has been inconclusive. Droplet digital PCR (ddPCR) offers an improvement over the current qPCR methods through the absolute quantification of viral loads. We compared ddPCR and qPCR results for the quantification of CMV DNA in blood and urine specimens from 39 neonates with cCMV (21 symptomatic and 18 asymptomatic). There was no significant difference in blood CMV DNA loads measured by ddPCR and qPCR, with or without any clinical findings. However, developmental delays at 36 months were significantly more frequently observed in patients with high CMV DNA loads (≥2950 copies/ml), as measured by ddPCR at diagnosis, than in those with lower CMV DNA loads. The association of urine CMV DNA load with symptoms and developmental delay was not observed. CMV DNA loads in the blood might be used as a predictor of developmental outcomes in cCMV patients, and absolute quantitation of viral loads by ddPCR assay could contribute to the standardization of CMV load measurement.

A cell-free DNA metagenomic sequencing assay that integrates the host injury response to infection.

High-throughput metagenomic sequencing offers an unbiased approach to identify pathogens in clinical samples. Conventional metagenomic sequencing, however, does not integrate information about the host, which is often critical to distinguish infection from infectious disease, and to assess the severity of disease. Here, we explore the utility of high-throughput sequencing of cell-free DNA (cfDNA) after bisulfite conversion to map the tissue and cell types of origin of host-derived cfDNA, and to profile the bacterial and viral metagenome. We applied this assay to 51 urinary cfDNA isolates collected from a cohort of kidney transplant recipients with and without bacterial and viral infection of the urinary tract. We find that the cell and tissue types of origin of urinary cfDNA can be derived from its genome-wide profile of methylation marks, and strongly depend on infection status. We find evidence of kidney and bladder tissue damage due to viral and bacterial infection, respectively, and of the recruitment of neutrophils to the urinary tract during infection. Through direct comparison to conventional metagenomic sequencing as well as clinical tests of infection, we find this assay accurately captures the bacterial and viral composition of the sample. The assay presented here is straightforward to implement, offers a systems view into bacterial and viral infections of the urinary tract, and can find future use as a tool for the differential diagnosis of infection.

Elimination of human cytomegalovirus DNA degradation in urine.

Congenital cytomegalovirus infection (cCMVi) is an important cause of sensorineural hearing loss in newborns. Detection of human cytomegalovirus (HCMV) DNA in urine has been used to screen for cCMVi in newborns. However, the matrix effect of urine on HCMV DNA detection is unclear. To evaluate the matrix effect of urine on HCMV DNA detection and optimize the sample process strategy to eliminate or minimize the impact of urine on HCMV DNA detection, DNA in spiked samples was extracted using different DNA extraction methods, and urine samples that could inhibit HCMV DNA detection were mixed to evaluate the inhibitory substances, inhibitory mechanism, and elimination of the inhibitory effect. The optimal urine sample process strategy was evaluated using 42 adult female urine samples and 42 newborn urine samples spiked with HCMV. Some urine samples were found to inhibit HCMV DNA detection due to DNA degradation. The addition of ≥5 mM EDTA to the urine before extraction eliminated the inhibitory effect of urine and did not affect the detection results of urine exhibiting no inhibition. Of the 42 adult female and 42 newborn urine samples, four and two samples, respectively, could inhibit HCMV DNA detection. However, the inhibitory effects of these six urine samples were eliminated after the addition of EDTA. The collective results indicate that the addition of EDTA can completely eliminate the impact of inhibitors present in urine on HCMV DNA extraction and improve the detection of HCMV in urine.

Epidemiology of cytomegalovirus Infection among mothers and infants in Colombia.

We assessed maternal and infant cytomegalovirus (CMV) infection in Colombia. Maternal serum was tested for CMV immunoglobulin G antibodies at a median of 10 (interquartile range: 8-12) weeks gestation (n = 1501). CMV DNA polymerase chain reaction was performed on infant urine to diagnose congenital (≤21 days of life) and postnatal (>21 days) infection. Maternal CMV seroprevalence was 98.1% (95% confidence interval [CI]: 97.5%-98.8%). Congenital CMV prevalence was 8.4 (95% CI: 3.9%-18.3%; 6/711) per 1000 live births. Among 472 infants without confirmed congenital CMV infection subsequently tested at age 6 months, 258 (54.7%, 95% CI: 50.2%-59.1%) had postnatal infection.

Prevalence and genotype distribution of JC polyomavirus in urine from patients with hematological malignancies in Vietnam.

JC virus (JCV) causes progressive multifocal leukoencephalopathy in immunocompromised patients. The prevalence and genotype patterns of JCV vary between different geographical regions. This study was done to investigate the prevalence and genotype distribution of JCV in patients with hematological malignancies in Vietnam. A total of 48 urine samples were collected from patients with hematological malignancies. DNA was extracted and detection of JCV was by nested-polymerase chain reaction. Sequence analysis was obtained and a phylogenetic tree was constructed for genotyping of JCV. Twenty-seven (56.25%) urine samples tested positive for JCV. JCV genotype 7 was only observed in this study. Subtype analysis showed that JCV subtype 7A was the most commonly prevalent, followed by 7B1 and 7C1. Other subtypes were not detected in this population. There were no significant differences associated with age, gender, and biochemical parameters between patients with JCV and without JCV excretion in urine. The present study showed a high prevalence of JCV in the urine of patients with hematologic malignancies. The most common genotype found in this population was JCV subtype 7A.

HPyV6 and HPyV7 in urine from immunocompromised patients.

BACKGROUND: Human polyomavirus 6 (HPyV6) and HPyV7 are two of the novel polyomaviruses that were originally detected in non-diseased skin. Serological studies have shown that these viruses are ubiquitous in the healthy adult population with seroprevalence up to 88% for HPyV6 and 72% for HPyV7. Both viruses are associated with pruritic skin eruption in immunocompromised patients, but a role with other diseases in immunoincompetent patients or malignancies has not been established. METHODS: PCR was used to determine the presence of HPyV6 and HPyV7 DNA in urine samples from systemic lupus erythematosus (n = 73), multiple sclerosis (n = 50), psoriasis vulgaris (n = 15), arthritic psoriasis (n = 15) and HIV-positive patients (n = 66). In addition, urine from pregnant women (n = 47) and healthy blood donors (n = 20) was investigated. RESULTS: HPyV6 DNA was detected in 21 (28.8%) of the urine specimens from SLE patients, in 6 (9.1%) of the urine samples from the HIV-positive cohort, and in 19 (40.4%) samples from pregnant women. HPyV7 DNA was only found in 6 (8.2%) of the urine specimens from SLE patients and in 4 (8.5%) samples from pregnant women. No HPyV6 and HPyV7 viruria was detected in the urine samples from the other patients. CONCLUSIONS: HPyV6, and to a lesser extend HPyV7, viruria seems to be common in SLE and HIV-positive patients, and pregnant women. Whether these viruses are of clinical relevance in these patients is not known.

The Yield of Targeted Examination for the Detection of Symptomatic Congenital Cytomegalovirus Infection.

BACKGROUND: The incidence of congenital cytomegalovirus (CMV) infection in Israel is 0.7%. Only 10-15% are symptomatic. Valganciclovir has been shown to improve hearing and neurodevelopmental outcomes in neonates with symptomatic congenital CMV infection. Targeted examination of infants who fail routine neonatal hearing screening or have clinical or laboratory findings suggestive of symptomatic congenital CMV infection may be a cost-effective approach. OBJECTIVES: To assess the possibility of targeted examination for the detection of newborns with symptomatic congenital CMV infection. METHODS: A prospective observational study was conducted in 2014-2015 at two medical centers in northern Israel. Included were all newborns who were tested in the first 3 days of life by polymerase chain reaction (PCR) for urine CMV DNA (n=692), either for failure the hearing screening (n=539, 78%), clinical or laboratory findings suggestive of symptomatic congenital CMV infection, or primary CMV infection during pregnancy (n=153, 22%). RESULTS: During the study period 15,433 newborns were born. The predicted rate of infection was 10-15% (symptomatic) of 0.7% of newborns, namely 0.07-0.105% or 10-15 infants. In fact, 15 infants (0.11%, 95% confidence interval 0.066-0.175) were diagnosed with symptomatic congenital CMV infection, 2/539 (0.37%) in the failed hearing group and 13/153 (8%) in the clinical/laboratory findings group. The incidence of symptomatic congenital CMV infection was within the predicted range. CONCLUSIONS: Targeted examination of only 4.5% (n=692) of newborns detected the predicted number of infants with symptomatic congenital CMV infection in whom valganciclovir therapy is recommended.

Impact of Collection Volume and DNA Extraction Method on the Detection of Biomarkers and HPV DNA in First-Void Urine.

The potential of first-void (FV) urine as a non-invasive liquid biopsy for detection of human papillomavirus (HPV) DNA and other biomarkers has been increasingly recognized over the past decade. In this study, we investigated whether the volume of this initial urine stream has an impact on the analytical performance of biomarkers. In parallel, we evaluated different DNA extraction protocols and introduced an internal control in the urine preservative. Twenty-five women, diagnosed with high-risk HPV, provided three home-collected FV urine samples using three FV urine collection devices (Colli-Pee) with collector tubes that differ in volume (4, 10, 20 mL). Each collector tube was prefilled with Urine Conservation Medium spiked with phocine herpesvirus 1 (PhHV-1) DNA as internal control. Five different DNA extraction protocols were compared, followed by PCR for GAPDH and PhHV-1 (qPCR), HPV DNA, and HBB (HPV-Risk Assay), and ACTB (methylation-specific qPCR). Results showed limited effects of collection volume on human and HPV DNA endpoints. In contrast, significant variations in yield for human endpoints were observed for different DNA extraction methods (p < 0.05). Additionally, the potential of PhHV-1 as internal control to monitor FV urine collection, storage, and processing was demonstrated.

Test Accuracy of Human Papillomavirus in Urine for Detection of Cervical Intraepithelial Neoplasia.

The objective was to assess the diagnostic test accuracy of high-risk human papillomavirus (hrHPV) testing of self-collected urine and cervicovaginal samples for the detection of cervical intraepithelial neoplasia grade 2 or higher (CIN2+). We recruited a convenience sample of women 25 to 65 years of age who were undergoing clinically indicated colposcopy at two medical centers in North Carolina between November 2016 and January 2019. Women with normal cytology results and positive hrHPV results were also recruited. Urine samples, self-collected cervicovaginal samples, provider-collected cervical samples, and cervical biopsy samples were obtained from all enrolled women. Samples were tested for hrHPV DNA using the Onclarity assay (Becton Dickinson, Sparks, MD). Biopsy samples were histologically graded as CIN2+ or

Incidence and risk factors for high-level BK viruria: a single center study in China.

BACKGROUND: BK virus allograft nephropathy is a serious complication after kidney transplantation, and the effect of pre-emptive intervention for high-level BK viruria has been verified, but protocols after kidney transplantation for early identification of high-level viruria are lacking. METHODS: This was a single-center study. The clinical data of the kidney transplant recipients and their donors in our center from January 1, 2015 to December 31, 2018, were collected. The patients were divided into the high-level BK viruria group (Group A) and a non-high-level BK viruria group (Group B) according to the qPCR results of BK virus DNA loads in urine samples. Significant variables were screened out by univariate analysis, and then the results were incorporated into a multivariate logistic regression model to analyze the independent risk factors for high-level BK viruria. RESULTS: A total of 262 recipients were included in the study. The incidence of high-level BK viruria was 13.4% (n = 35), and the median time of detection was 181 (range 91-1119) days. Univariate analysis showed that donor type ([Formula: see text] = 21.770, P < 0.001), history of ATG/ATG-F application ([Formula: see text] = 4.543, P = 0.033), acute rejection (AR) ([Formula: see text] = 8.313, P = 0.004) and delayed graft function (DGF) ([Formula: see text] = 21.170, P < 0.001) were related to high-level BK viruria. After the inclusion of the multivariate logistic regression model, the results showed deceased brain and cardiac donors (P = 0.032, OR = 3.927, 95% CI 1.122-13.746), AR (P = 0.022, OR = 4.709, 95% CI 1.253-17.697) and DGF (P = 0.001, OR = 6.682, 95% CI 2.288-19.518). CONCLUSIONS: Donation by deceased brain and cardiac patients, history of AR and DGF were independent risk factors for high-level BK viruria after kidney transplantation.

Assessment of clinical performance of an ultrasensitive nanowire assay for detecting human papillomavirus DNA in urine.

OBJECTIVE: To evaluate whether HPV DNA in urine has potential advantages as an alternative biomarker for HPV-based cervical cancer screening. METHODS: Among patients with Cobas HPV test results, a total of 67 HPV-positive (n = 42) and -negative (n = 25) women who agreed to participate in this study were willing to provide paired cervical and urine samples, and we observed concordance between sample types from each patient in identifying HPV genotypes using the nanowire assay. RESULTS: We detected high-risk strains of HPV DNA in unprocessed urine specimens using polyethyleneimine-conjugated nanowires (PEI-NWs). Concordance for high-risk HPV (hrHPV) between paired urine and cervical samples was 90.4% (κ = 0.90; 95% CI: 0.80-100.00). The virological sensitivity and specificity for detection of HPV DNA from a small urine sample (200 µL) were 81.3% (κ = 0.83; 95% CI: 62.1-100.0) and 98.0% (κ = 0.83; 95% CI: 94.2-100.0) for HPV16 group, 100.0% (κ = 0.65; 95% CI: 100.0-100.0) and 95.3% (κ = 0.65; 95% CI: 90.1-100.0) for HPV18 group, and 96.4% (κ = 0.97; 95% CI: 89.6-100.0) and 100.0% (κ = 0.97; 95% CI: 100.0-100.0) for other hrHPV group, respectively. CONCLUSIONS: The nanowire assay demonstrated excellent ability to identify HPV DNA from urine specimens. We observed an excellent agreement in the detection of high-risk HPV between paired urine and cervical samples, even with small urine sample volume.

A cohort study of the universal neonatal urine screening for congenital cytomegalovirus infection.

OBJECTIVES: This prospective cohort study aimed to evaluate the efficacy of the universal neonatal urine screening, followed by diagnosis, workup and antiviral therapy for symptomatic congenital cytomegalovirus (CMV) infection to reduce neurological impairments and sequelae. METHODS: Neonates born in three facilities underwent the universal urine screening of PCR analyses for CMV-DNA. Neonates with symptomatic congenital CMV infection (cCMV) received oral valganciclovir (VGCV) of 32 mg/kg/day for six weeks or six months, and were evaluated for neurological outcomes including developmental quotient (DQ) and hearing function at around 18 months of corrected age. RESULTS: cCMV was diagnosed in 56 (0.48%) of 11,736 neonates, consisting of 23 neonates with symptomatic and 33 with asymptomatic cCMV. The incidence of cCMV in the general perinatal medical center (0.69%) was higher than that in the primary maternity hospital (0.23%, p<0.01%). Twenty of the 23 infants with symptomatic cCMV received VGCV therapy, and 19 underwent neurological assessment. Eight neonates (42%) had severe sequelae of DQ < 70, bilateral hearing dysfunction, and/or epilepsy. Four neonates (21%) had mild sequelae of DQ 70-79 or unilateral hearing dysfunction only, and seven (37%) showed normal development without any impairment. CONCLUSIONS: This study on a large scale demonstrated that a series of universal neonatal urine screening, diagnosis, workup, and VGCV therapy for neonates with symptomatic cCMV may decrease neurological impairments, because 58% of the treated infants had normal development or mild sequelae. The universal urine screening likely identifies subclinical symptomatic cCMV. Mothers with fetuses of cCMV seem to be selectively transferred to perinatal medical centers before deliveries.

High prevalence of breastmilk-acquired cytomegalovirus infection in jaundiced infants.

BACKGROUND: Our objective was to evaluate the prevalence and different diagnostic methods of breastmilk (BM)-acquired cytomegalovirus (CMV) infection in a pathologically jaundiced cohort. METHODS: A total of 400 infants confirmed with pathological jaundice at The People's Hospital of Qingyang City were screened for BM-acquired CMV infection between February 2018 and February 2019. A total of 300 infants were finally enrolled in our study. CMV infection was confirmed by detecting both CMV-DNA in various samples using FQ-PCR and CMV-IgM with chemiluminescence. Clinical and other laboratory data were collected from these infants during their hospitalization or regular visits. RESULTS: Ninety-eight (32.67%) subjects were confirmed to be BM CMV-DNA-positive, and 18 (18.37%) were diagnosed with a BM-acquired CMV infection. All 18 (100%) infants with a BM-acquired CMV infection were CMV-DNA-positive in urine, while 5 (27.78%) cases and 11 (61.11%) cases were confirmed in plasma and peripheral blood mononuclear cells (PBMCs), respectively. Only 6 (33.33%) infants were CMV-IgM-positive. Birthweight, direct bilirubin, aspartate aminotransferase, and the viral load in BM of the BM-acquired CMV group were higher than those in the non-infected group (P < .05). Low birthweight and viral load in BM were risk factors for BM-acquired CMV infection. Detecting CMV-DNA in urine samples exhibited better performance than the other methods for screening BM-acquired CMV infections. CONCLUSIONS: Our study found a high prevalence of BM-acquired CMV infection in jaundiced infants, and detecting CMV-DNA in a urine sample was the most sensitive method for disease screening.

Analysis of the Pharmacokinetics of Suppository Forms of Bacteriophages.

Pharmacokinetics of suppository forms of bacteriophages was studied on male Chinchilla rabbits. Suppositories with various composition of bacteriophages were administered once per rectum to rabbits, and the presence of phage particles was estimated in the blood, urine, and feces over 24 h. Pharmacokinetic study showed that the phages were detected in the blood, urine, and feces at various terms of the experiment irrespective of the size of viral particles, which confirmed the possibility of their systemic effects after rectal administration. Thus, the use of suppository form of bacteriophages can ensure the presence of phage particles even in infection foci that cannot directly contact with the preparation.

Cobalt Oxide Porous Nanocubes-Based Electrochemical Immunobiosensing of Hepatitis B Virus DNA in Blood Serum and Urine Samples.

In this work, we report a new biosensing platform for hepatitis B virus (HBV) DNA genosensing using cobalt oxide (Co3O4) nanostructures. The tunable morphologies of Co3O4 nanostructures such as porous nanocubes (PNCs), nanooctahedra (NOHs), and nanosticks (NSKs) are synthesized, and characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD) patterns, nitrogen adsorption/desorption isotherms (BET), and electrochemical impedance spectral (EIS) methods. The HBV probe DNA (ssDNA) is immobilized on the Co3O4 nanostructures through coordinate bond formation between nucleic acid of ssDNA and Co metal, which results in highly stable nanostructured biosensing platform. To the best of our knowledge, first time the target cDNA of HBV is detected using ssDNA/Co3O4PNCs/GCE electrode by EIS method with a limit of detection (LOD) of 0.38 pM (signal-to-noise ratio (S/N) = 3). Moreover, the ssDNA/Co3O4PNCs/GCE has shown excellent specificity to HBV target cDNA, compared with noncomplementary DNA, and 1- and 3-mismatch DNAs. Finally, we explore ssDNA/Co3O4PNCs/GCE as potential electrode to test HBV DNA in blood serum and urine samples for practical applications.

Investigation of Preanalytical Variables Impacting Pathogen Cell-Free DNA in Blood and Urine.

Pathogen cell-free DNA (pcfDNA) in blood and urine is an attractive biomarker; however, the impact of preanalytical factors is not well understood. Blood and urine samples from healthy donors spiked with cfDNA from Mycobacterium tuberculosis, Salmonella enterica, Aspergillus fumigatus, and Epstein-Barr virus (EBV) and samples from tuberculosis patients were used to evaluate the impact of blood collection tube, urine preservative, processing delay, processing method, freezing and thawing, and sample volume on pcfDNA. The PCR cycle threshold (CT ) was used to measure amplifiable cfDNA. In spiked samples, the median CT values for M. tuberculosis, S. enterica, and EBV cfDNA were significantly lower in blood collected in K2EDTA tubes than those in Streck and PAXgene blood collection tubes, and they were was significantly lower in urine preserved with EDTA (EDTA-urine) than in urine preserved with Streck reagent (Streck-urine). Blood and urine samples from TB patients preserved with K2EDTA and Tris-EDTA, respectively, showed significantly lower median M. tuberculosisCT values than with the Streck blood collection tube and Streck urine preservative. Processing delay increased the median pathogen CT values for Streck and PAXgene but not K2EDTA blood samples and for urine preserved with Streck reagent but not EDTA. Double-spin compared with single-spin plasma separation increased the median pathogen CT regardless of blood collection tube. No differences were observed between whole urine and supernatant and between fresh and thawed plasma and urine after 24 weeks at -80°C. Larger plasma and urine volumes in contrived and patient samples showed a significantly lower median M. tuberculosisCT These findings suggest that large-volume single-spin K2EDTA-plasma and EDTA-whole urine with up to a 24-h processing delay may optimize pcfDNA detection.

JC virus identified in a patient with persistent and severe West Nile virus disease.

West Nile virus is a notable cause of neuroinvasive disease, damage to the central nervous system, or even death. In this study, using metagenomics analysis and quantitative real-time PCR validation, we identified a JC virus infection in urine and cerebrospinal fluid samples of a West Nile virus patient with severe neurological symptoms and extended disease. JC virus is known to be involved in neurological complications, especially in immunocompromised individuals thus suggesting that the coinfection with JC virus is involved with the West Nile virus infection persistence and severe symptoms. JC virus was identified in urine samples from additional West Nile virus patients via quantitative real-time PCR, however, JC virus was not found in any cerebrospinal fluid samples of West Nile virus patients, suggesting that JC virus does not regularly infect the central nervous system of WNV patients. Overall, this study highlights the importance of identifying infection by opportunistic viruses in already-diagnosed patients and highlights the advantages of next-generation sequencing and metagenomics for viral diagnosis.

Progressive multifocal leukoencephalopathy in Zambia is caused by JC virus with prototype regulatory region.

There are only few documented cases of progressive multifocal leukoencephalopathy (PML) in Africa. Whether this is caused by a lack of JC virus (JCV) spread or alteration in the JCV genome is unknown. We characterized the clinical presentation, laboratory findings, and JCV regulatory region (RR) pattern of the first documented PML cases in Zambia as well as JCV seroprevalence among HIV+ and HIV- Zambians. We identified PML patients with positive JCV DNA PCR in their cerebrospinal fluid (CSF) among subjects enrolled in an ongoing tuberculous meningitis study from 2014 to 2016 in Lusaka. JCV regulatory region was further characterized by duplex PCR in patients' urine and CSF. Of 440 HIV+ patients, 14 (3%) had detectable JCV DNA in their CSF (age 18-50; CD4+ T cells counts 15-155 × 106/µl) vs 0/60 HIV- patients. The main clinical manifestations included altered mental status and impaired consciousness consistent with advanced PML. While prototype JCV was identified by duplex PCR assay in the CSF samples of all 14 PML patients, only archetype JCV was detected in their urine. All PML Zambian patients tested were seropositive for JCV compared to 46% in a control group of HIV+ and HIV- Zambian patients without PML. PML occurs among HIV-infected individuals in Zambia and is caused by CNS infection with prototype JCV, while archetype JCV strains are present in their urine. JCV seroprevalence is comparable in Zambia and the USA, and PML should be included in the differential diagnosis of immunosuppressed individuals presenting with neurological dysfunction in Zambia.