Chapare Hemorrhagic Fever and Virus Detection in Rodents in Bolivia in 2019.

BACKGROUND: In June 2019, the Bolivian Ministry of Health reported a cluster of cases of hemorrhagic fever that started in the municipality of Caranavi and expanded to La Paz. The cause of these cases was unknown. METHODS: We obtained samples for next-generation sequencing and virus isolation. Human and rodent specimens were tested by means of virus-specific real-time quantitative reverse-transcriptase-polymerase-chain-reaction assays, next-generation sequencing, and virus isolation. RESULTS: Nine cases of hemorrhagic fever were identified; four of the patients with this illness died. The etiologic agent was identified as Mammarenavirus Chapare mammarenavirus, or Chapare virus (CHAPV), which causes Chapare hemorrhagic fever (CHHF). Probable nosocomial transmission among health care workers was identified. Some patients with CHHF had neurologic manifestations, and those who survived had a prolonged recovery period. CHAPV RNA was detected in a variety of human body fluids (including blood; urine; nasopharyngeal, oropharyngeal, and bronchoalveolar-lavage fluid; conjunctiva; and semen) and in specimens obtained from captured small-eared pygmy rice rats (Oligoryzomys microtis). In survivors of CHHF, viral RNA was detected up to 170 days after symptom onset; CHAPV was isolated from a semen sample obtained 86 days after symptom onset. CONCLUSIONS: M. Chapare mammarenavirus was identified as the etiologic agent of CHHF. Both spillover from a zoonotic reservoir and possible person-to-person transmission were identified. This virus was detected in a rodent species, O. microtis. (Funded by the Bolivian Ministry of Health and others.).

Risk Factors for Ebola Virus Persistence in Semen of Survivors in Liberia.

BACKGROUND: Long-term persistence of Ebola virus (EBOV) in immunologically privileged sites has been implicated in recent outbreaks of Ebola virus disease (EVD) in Guinea and the Democratic Republic of Congo. This study was designed to understand how the acute course of EVD, convalescence, and host immune and genetic factors may play a role in prolonged viral persistence in semen. METHODS: A cohort of 131 male EVD survivors in Liberia were enrolled in a case-case study. "Early clearers" were defined as those with 2 consecutive negative EBOV semen test results by real-time reverse-transcription polymerase chain reaction (rRT-PCR) ≥2 weeks apart within 1 year after discharge from the Ebola treatment unit or acute EVD. "Late clearers" had detectable EBOV RNA by rRT-PCR >1 year after discharge from the Ebola treatment unit or acute EVD. Retrospective histories of their EVD clinical course were collected by questionnaire, followed by complete physical examinations and blood work. RESULTS: Compared with early clearers, late clearers were older (median, 42.5 years; P < .001) and experienced fewer severe clinical symptoms (median 2, P = .006). Late clearers had more lens opacifications (odds ratio, 3.9 [95% confidence interval, 1.1-13.3]; P = .03), after accounting for age, higher total serum immunoglobulin G3 (IgG3) titers (P = .005), and increased expression of the HLA-C*03:04 allele (0.14 [.02-.70]; P = .007). CONCLUSIONS: Older age, decreased illness severity, elevated total serum IgG3 and HLA-C*03:04 allele expression may be risk factors for the persistence of EBOV in the semen of EVD survivors. EBOV persistence in semen may also be associated with its persistence in other immunologically protected sites, such as the eye.

Remdesivir targets a structurally analogous region of the Ebola virus and SARS-CoV-2 polymerases.

Remdesivir is a broad-spectrum antiviral nucleotide prodrug that has been clinically evaluated in Ebola virus patients and recently received emergency use authorization (EUA) for treatment of COVID-19. With approvals from the Federal Select Agent Program and the Centers for Disease Control and Prevention's Institutional Biosecurity Board, we characterized the resistance profile of remdesivir by serially passaging Ebola virus under remdesivir selection; we generated lineages with low-level reduced susceptibility to remdesivir after 35 passages. We found that a single amino acid substitution, F548S, in the Ebola virus polymerase conferred low-level reduced susceptibility to remdesivir. The F548 residue is highly conserved in filoviruses but should be subject to specific surveillance among novel filoviruses, in newly emerging variants in ongoing outbreaks, and also in Ebola virus patients undergoing remdesivir therapy. Homology modeling suggests that the Ebola virus polymerase F548 residue lies in the F-motif of the polymerase active site, a region that was previously identified as susceptible to resistance mutations in coronaviruses. Our data suggest that molecular surveillance of this region of the polymerase in remdesivir-treated COVID-19 patients is also warranted.

Lassa Virus Replicon Particle Vaccine Protects Strain 13/N Guinea Pigs Against Challenge With Geographically and Genetically Diverse Viral Strains.

Lassa virus (LASV) causes mild to severe hemorrhagic fever disease in humans. Strain 13/N guinea pigs are highly susceptible to infection with LASV strain Josiah (clade IV), providing a critical model system for therapeutics and vaccine development. To develop additional models of disease, we detail the clinical course in guinea pigs infected with 5 geographically and genetically diverse LASV strains. Two of the developed models (LASV clades II and III) were then used to evaluate efficacy of a virus replicon particle vaccine against heterologous LASV challenge, demonstrating complete protection against clinical disease after a single vaccination dose.

Transmission of Nipah Virus - 14 Years of Investigations in Bangladesh.

BACKGROUND: Nipah virus is a highly virulent zoonotic pathogen that can be transmitted between humans. Understanding the dynamics of person-to-person transmission is key to designing effective interventions. METHODS: We used data from all Nipah virus cases identified during outbreak investigations in Bangladesh from April 2001 through April 2014 to investigate case-patient characteristics associated with onward transmission and factors associated with the risk of infection among patient contacts. RESULTS: Of 248 Nipah virus cases identified, 82 were caused by person-to-person transmission, corresponding to a reproduction number (i.e., the average number of secondary cases per case patient) of 0.33 (95% confidence interval [CI], 0.19 to 0.59). The predicted reproduction number increased with the case patient's age and was highest among patients 45 years of age or older who had difficulty breathing (1.1; 95% CI, 0.4 to 3.2). Case patients who did not have difficulty breathing infected 0.05 times as many contacts (95% CI, 0.01 to 0.3) as other case patients did. Serologic testing of 1863 asymptomatic contacts revealed no infections. Spouses of case patients were more often infected (8 of 56 [14%]) than other close family members (7 of 547 [1.3%]) or other contacts (18 of 1996 [0.9%]). The risk of infection increased with increased duration of exposure of the contacts (adjusted odds ratio for exposure of >48 hours vs. ≤1 hour, 13; 95% CI, 2.6 to 62) and with exposure to body fluids (adjusted odds ratio, 4.3; 95% CI, 1.6 to 11). CONCLUSIONS: Increasing age and respiratory symptoms were indicators of infectivity of Nipah virus. Interventions to control person-to-person transmission should aim to reduce exposure to body fluids. (Funded by the National Institutes of Health and others.).

Characteristics of Ebola Virus Disease Survivor Blood and Semen in Liberia: Serology and Reverse Transcription Polymerase Chain Reaction (RT-PCR).

INTRODUCTION: Ebola virus (EBOV), species Zaire ebolavirus, may persist in the semen of male survivors of Ebola virus disease (EVD). We conducted a study of male survivors of the 2014-2016 EVD outbreak in Liberia and evaluated their immune responses to EBOV. We report here findings from the serologic testing of blood for EBOV-specific antibodies, molecular testing for EBOV in blood and semen, and serologic testing of peripheral blood mononuclear cells (PBMCs) in a subset of study participants. METHODS: We tested for EBOV RNA in blood by quantitative reverse transcription polymerase chain reaction (qRT-PCR), and for anti-EBOV-specific immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies by enzyme-linked immunosorbent assay (ELISA) for 126 study participants. We performed PBMC analysis on a subgroup of 26 IgG-negative participants. RESULTS: All 126 participants tested negative for EBOV RNA in blood by qRT-PCR. The blood of 26 participants tested negative for EBOV-specific IgG antibodies by ELISA. PBMCs were collected from 23/26 EBOV IgG-negative participants. Of these, 1/23 participants had PBMCs that produced anti-EBOV-specific IgG antibodies upon stimulation with EBOV-specific glycoprotein (GP) and nucleoprotein (NP) antigens. CONCLUSIONS: The blood of EVD survivors, collected when they did not have symptoms meeting the case definition for acute or relapsed EVD, is unlikely to pose a risk for EBOV transmission. We identified 1 IgM/IgG negative participant who had PBMCs that produced anti-EBOV-specific antibodies upon stimulation. Immunogenicity following acute EBOV infection may exist along a spectrum, and absence of antibody response should not be exclusionary in determining an individual's status as a survivor of EVD.

Fluorescent Crimean-Congo hemorrhagic fever virus illuminates tissue tropism patterns and identifies early mononuclear phagocytic cell targets in Ifnar-/- mice.

Crimean-Congo hemorrhagic fever virus (CCHFV, order Bunyavirales, family Nairoviridae, genus Orthonairovirus) is the tick-borne etiological agent of Crimean-Congo hemorrhagic fever (CCHF) in humans. Animals are generally susceptible to CCHFV infection but refractory to disease. Small animal models are limited to interferon-deficient mice, that develop acute fatal disease following infection. Here, using a ZsGreen1- (ZsG) expressing reporter virus (CCHFV/ZsG), we examine tissue tropism and dissemination of virus in interferon-α/ß receptor knock-out (Ifnar-/-) mice. We demonstrate that CCHFV/ZsG retains in vivo pathogenicity comparable to wild-type virus. Interestingly, despite high levels of viral RNA in all organs assessed, 2 distribution patterns of infection were observed by both fluorescence and immunohistochemistry (IHC), corresponding to the permissiveness of organ tissues. To further investigate viral dissemination and to temporally define cellular targets of CCHFV in vivo, mice were serially euthanized at different stages of disease. Flow cytometry was used to characterize CCHFV-associated alterations in hematopoietic cell populations and to classify infected cells in the blood, lymph node, spleen, and liver. ZsG signal indicated that mononuclear phagocytic cells in the lymphatic tissues were early targets of infection; in late-stage infection, overall, the highest levels of signal were detected in the liver, and ZsG was found in both antigen-presenting and lymphocyte cell populations.

Inhibition of Nipah Virus by Defective Interfering Particles.

The error-prone nature of RNA-dependent RNA polymerases drives the diversity of RNA virus populations. Arising within this diversity is a subset of defective viral genomes that retain replication competency, termed defective interfering (DI) genomes. These defects are caused by aberrant viral polymerase reinitiation on the same viral RNA template (deletion DI species) or the nascent RNA strand (copyback DI species). DI genomes have previously been shown to alter the dynamics of a viral population by interfering with normal virus replication and/or by stimulating the innate immune response. In this study, we investigated the ability of artificially produced DI genomes to inhibit Nipah virus (NiV), a highly pathogenic biosafety level 4 paramyxovirus. High multiplicity of infection passaging of both NiV clinical isolates and recombinant NiV in Vero cells generated an extensive DI population from which individual DIs were identified using next-generation sequencing techniques. Assays were established to generate and purify both naturally occurring and in silico-designed DIs as fully encapsidated, infectious virus-like particles termed defective interfering particles (DIPs). We demonstrate that several of these NiV DIP candidates reduced NiV titers by up to 4 logs in vitro. These data represent a proof-of-principle that a therapeutic application of DIPs to combat NiV infections may be an alternative source of antiviral control for this disease.

In Situ Imaging of Fluorescent Nipah Virus Respiratory and Neurological Tissue Tropism in the Syrian Hamster Model.

Using a recombinant Nipah virus expressing a fluorescent protein (ZsG), we visualized virus tropism in the Syrian hamster model. We found that anatomical localization of fluorescence correlated to clinical signs; signal was primarily visualized in the respiratory tract in animals with acute-onset terminal disease, whereas central nervous system localization was seen in animals that succumbed with delayed disease onset. While polymerase chain reaction (PCR) detection corresponded well to ZsG signal, virus was only isolated from some lung, brain, liver, and kidney samples that were ZsG and/or PCR positive, and only from animals euthanized on or before 15 days post infection.

Alterations in Blood Chemistry Levels Associated With Nipah Virus Disease in the Syrian Hamster Model.

Nipah virus (NiV; family Paramyxoviridae, genus Henipavirus) infection can cause severe respiratory and neurological disease in humans. The pathophysiology of disease is not fully understood, and it may vary by presentation and clinical course. In this study, we investigate changes in blood chemistry in NiV-infected Syrian hamsters that survived or succumbed to disease. Increased sodium and magnesium and decreased albumin and lactate levels were detected in animals euthanized with severe clinical disease compared with mock-infected controls. When subjects were grouped by clinical syndrome, additional trends were discernable, highlighting changes associated with either respiratory or neurological disease.

Changing Contact Patterns Over Disease Progression: Nipah Virus as a Case Study.

Contact patterns play a key role in disease transmission, and variation in contacts during the course of illness can influence transmission, particularly when accompanied by changes in host infectiousness. We used surveys among 1642 contacts of 94 Nipah virus case patients in Bangladesh to determine how contact patterns (physical and with bodily fluids) changed as disease progressed in severity. The number of contacts increased with severity and, for case patients who died, peaked on the day of death. Given transmission has only been observed among fatal cases of Nipah virus infection, our findings suggest that changes in contact patterns during illness contribute to risk of infection.

Griffithsin Inhibits Nipah Virus Entry and Fusion and Can Protect Syrian Golden Hamsters From Lethal Nipah Virus Challenge.

Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus that causes fatal encephalitis and respiratory disease in humans. There is currently no approved therapeutic for human use against NiV infection. Griffithsin (GRFT) is high-mannose oligosaccharide binding lectin that has shown in vivo broad-spectrum activity against viruses, including severe acute respiratory syndrome coronavirus, human immunodeficiency virus 1, hepatitis C virus, and Japanese encephalitis virus. In this study, we evaluated the in vitro antiviral activities of GRFT and its synthetic trimeric tandemer (3mG) against NiV and other viruses from 4 virus families. The 3mG had comparatively greater potency than GRFT against NiV due to its enhanced ability to block NiV glycoprotein-induced syncytia formation. Our initial in vivo prophylactic evaluation of an oxidation-resistant GRFT (Q-GRFT) showed significant protection against lethal NiV challenge in Syrian golden hamsters. Our results warrant further development of Q-GRFT and 3mG as potential NiV therapeutics.

Evaluation of a Single-Dose Nucleoside-Modified Messenger RNA Vaccine Encoding Hendra Virus-Soluble Glycoprotein Against Lethal Nipah virus Challenge in Syrian Hamsters.

In the absence of approved vaccines and therapeutics for use in humans, Nipah virus (NiV) continues to cause fatal outbreaks of encephalitis and respiratory disease in Bangladesh and India on a near-annual basis. We determined that a single dose of a lipid nanoparticle nucleoside-modified messenger RNA vaccine encoding the soluble Hendra virus glycoprotein protected up to 70% of Syrian hamsters from lethal NiV challenge, despite animals having suboptimally primed immune responses before challenge. These data provide a foundation from which to optimize future messenger RNA vaccination studies against NiV and other highly pathogenic viruses.

Seoul Virus Infection and Spread in United States Home-Based Ratteries: Rat and Human Testing Results From a Multistate Outbreak Investigation.

BACKGROUND: During 2017, a multistate outbreak investigation occurred after the confirmation of Seoul virus (SEOV) infections in people and pet rats. A total of 147 humans and 897 rats were tested. METHODS: In addition to immunoglobulin (Ig)G and IgM serology and traditional reverse-transcription polymerase chain reaction (RT-PCR), novel quantitative RT-PCR primers/probe were developed, and whole genome sequencing was performed. RESULTS: Seventeen people had SEOV IgM, indicating recent infection; 7 reported symptoms and 3 were hospitalized. All patients recovered. Thirty-one facilities in 11 US states had SEOV infection, and among those with ≥10 rats tested, rat IgG prevalence ranged 2%-70% and SEOV RT-PCR positivity ranged 0%-70%. Human laboratory-confirmed cases were significantly associated with rat IgG positivity and RT-PCR positivity (P = .03 and P = .006, respectively). Genomic sequencing identified >99.5% homology between SEOV sequences in this outbreak, and these were >99% identical to SEOV associated with previous pet rat infections in England, the Netherlands, and France. Frequent trade of rats between home-based ratteries contributed to transmission of SEOV between facilities. CONCLUSIONS: Pet rat owners, breeders, and the healthcare and public health community should be aware and take steps to prevent SEOV transmission in pet rats and to humans. Biosecurity measures and diagnostic testing can prevent further infections.

Ebola RNA Persistence in Semen of Ebola Virus Disease Survivors - Final Report.

BACKGROUND: Ebola virus has been detected in the semen of men after their recovery from Ebola virus disease (EVD). We report the presence of Ebola virus RNA in semen in a cohort of survivors of EVD in Sierra Leone. METHODS: We enrolled a convenience sample of 220 adult male survivors of EVD in Sierra Leone, at various times after discharge from an Ebola treatment unit (ETU), in two phases (100 participants were in phase 1, and 120 in phase 2). Semen specimens obtained at baseline were tested by means of a quantitative reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assay with the use of the target sequences of NP and VP40 (in phase 1) or NP and GP (in phase 2). This study did not evaluate directly the risk of sexual transmission of EVD. RESULTS: Of 210 participants who provided an initial semen specimen for analysis, 57 (27%) had positive results on quantitative RT-PCR. Ebola virus RNA was detected in the semen of all 7 men with a specimen obtained within 3 months after ETU discharge, in 26 of 42 (62%) with a specimen obtained at 4 to 6 months, in 15 of 60 (25%) with a specimen obtained at 7 to 9 months, in 4 of 26 (15%) with a specimen obtained at 10 to 12 months, in 4 of 38 (11%) with a specimen obtained at 13 to 15 months, in 1 of 25 (4%) with a specimen obtained at 16 to 18 months, and in no men with a specimen obtained at 19 months or later. Among the 46 participants with a positive result in phase 1, the median baseline cycle-threshold values (higher values indicate lower RNA values) for the NP and VP40 targets were lower within 3 months after ETU discharge (32.4 and 31.3, respectively; in 7 men) than at 4 to 6 months (34.3 and 33.1; in 25), at 7 to 9 months (37.4 and 36.6; in 13), and at 10 to 12 months (37.7 and 36.9; in 1). In phase 2, a total of 11 participants had positive results for NP and GP targets (samples obtained at 4.1 to 15.7 months after ETU discharge); cycle-threshold values ranged from 32.7 to 38.0 for NP and from 31.1 to 37.7 for GP. CONCLUSIONS: These data showed the long-term presence of Ebola virus RNA in semen and declining persistence with increasing time after ETU discharge. (Funded by the World Health Organization and others.).

Characterization of Novel Reoviruses Wad Medani Virus (Orbivirus) and Kundal Virus (Coltivirus) Collected from Hyalomma anatolicum Ticks in India during Surveillance for Crimean Congo Hemorrhagic Fever.

In 2011, ticks were collected from livestock following an outbreak of Crimean Congo hemorrhagic fever (CCHF) in Gujarat state, India. CCHF-negative Hyalomma anatolicum tick pools were passaged for virus isolation, and two virus isolates were obtained, designated Karyana virus (KARYV) and Kundal virus (KUNDV), respectively. Traditional reverse transcription-PCR (RT-PCR) identification of known viruses was unsuccessful, but a next-generation sequencing (NGS) approach identified KARYV and KUNDV as viruses in the Reoviridae family, Orbivirus and Coltivirus genera, respectively. Viral genomes were de novo assembled, yielding 10 complete segments of KARYV and 12 nearly complete segments of KUNDV. The VP1 gene of KARYV shared a most recent common ancestor with Wad Medani virus (WMV), strain Ar495, and based on nucleotide identity we demonstrate that it is a novel WMV strain. The VP1 segment of KUNDV shares a common ancestor with Colorado tick fever virus, Eyach virus, Tai Forest reovirus, and Tarumizu tick virus from the Coltivirus genus. Based on VP1, VP6, VP7, and VP12 nucleotide and amino acid identities, KUNDV is proposed to be a new species of Coltivirus Electron microscopy supported the classification of KARYV and KUNDV as reoviruses and identified replication morphology consistent with other orbi- and coltiviruses. The identification of novel tick-borne viruses carried by the CCHF vector is an important step in the characterization of their potential role in human and animal pathogenesis.IMPORTANCE Ticks and mosquitoes, as well Culicoides, can transmit viruses in the Reoviridae family. With the help of next-generation sequencing (NGS), previously unreported reoviruses such as equine encephalosis virus, Wad Medani virus (WMV), Kammavanpettai virus (KVPTV), and, with this report, KARYV and KUNDV have been discovered and characterized in India. The isolation of KUNDV and KARYV from Hyalomma anatolicum, which is a known vector for zoonotic pathogens, such as Crimean Congo hemorrhagic fever virus, Babesia, Theileria, and Anaplasma species, identifies arboviruses with the potential to transmit to humans. Characterization of KUNDV and KARYV isolated from Hyalomma ticks is critical for the development of specific serological and molecular assays that can be used to determine the association of these viruses with disease in humans and livestock.

Antibody-Mediated Virus Neutralization Is Not a Universal Mechanism of Marburg, Ebola, or Sosuga Virus Clearance in Egyptian Rousette Bats.

Although bats are increasingly being recognized as natural reservoir hosts of emerging zoonotic viruses, little is known about how they control and clear virus infection in the absence of clinical disease. Here, we test >50 convalescent sera from Egyptian rousette bats (ERBs) experimentally primed or prime-boosted with Marburg virus, Ebola virus, or Sosuga virus for the presence of virus-specific neutralizing antibodies, using infectious reporter viruses. After serum neutralization testing, we conclude that antibody-mediated virus neutralization does not contribute significantly to the control and clearance of Marburg virus, Ebola virus, or Sosuga virus infection in ERBs.

Protection From Lethal Lassa Disease Can Be Achieved Both Before and After Virus Exposure by Administration of Single-Cycle Replicating Lassa Virus Replicon Particles.

Lassa fever is a frequently severe human disease that is endemic to several countries in West Africa. To date, no licensed vaccines are available to prevent Lassa virus (LASV) infection, even though Lassa fever is thought to be an important disease contributing to mortality and both acute and chronic morbidity. We have previously described a vaccine candidate composed of single-cycle LASV replicon particles (VRPs) and a stable cell line for their production. Here, we refine the genetic composition of the VRPs and demonstrate the ability to reproducibly purify them with high yields. Studies in the guinea pig model confirm efficacy of the vaccine candidate, demonstrate that single-cycle replication is necessary for complete protection by the VRP vaccine, and show that postexposure vaccination can confer protection from lethal outcome.

Adaptive Immune Responses in Humans During Nipah Virus Acute and Convalescent Phases of Infection.

BACKGROUND: Nipah virus (NiV) is 1 of 10 potential causes of imminent public health emergencies of international concern. We investigated the NiV outbreak that occurred in May 2018 in Kerala, India. Here we describe the longitudinal characteristics of cell-mediated and humoral immune responses to NiV infection during the acute and convalescent phases in 2 human survivors. METHODS: Serial blood samples were obtained from the only 2 survivors of the NiV outbreak in Kerala. We used flow cytometry to determine the absolute T-lymphocyte and B-lymphocyte counts and the phenotypes of both T and B cells. We also detected and quantitated the humoral immune response to NiV by virus-specific immunoglobulin M (IgM) and immunoglobulin G (IgG) enzyme-linked immunosorbent assay. RESULTS: Absolute numbers of T lymphocytes remained within normal limits throughout the period of illness studied in both survivors. However, a marked elevation of activated CD8 T cells was observed in both cases. More than 30% of total CD8 T cells expressed Ki67, indicating active proliferation. Proliferating (Ki-67+) CD8 T cells expressed high levels of granzyme B and PD-1, consistent with the profile of acute effector cells. Total B-lymphocyte, activated B-cell, and plasmablast counts were also elevated in NiV survivors. These individuals developed detectable NiV-specific IgM and IgG antibodies within a week of disease onset. Clearance of NiV RNA from blood preceded the appearance of virus-specific IgG and coincided with the peak of activated CD8 T cells. CONCLUSIONS: We describe for the first time longitudinal kinetic data on the activation status of human B- and T-cell populations during acute NiV infection. While marked CD8 T-cell activation was observed with effector characteristics, activated CD4 T cells were less prominent.

Suboptimal Handling of Piccolo Samples or Reagent Discs for Consideration in Ebola Response.

Operating clinical analyzers within recommended parameters can be challenging during outbreak response. Using the Piccolo Xpress point-of-care blood chemistry analyzer on guinea pig blood, we found that values of many analytes are still readily comparable when samples and reagent discs are handled at various conditions outside of manufacturer recommendations.