Human herpes virus type-6 is associated with central nervous system infections in children in Sudan.

Background: Human herpes virus type-6 (HHV-6) is increasingly recognised as a febrile agent in children. However, less is known in sub-Saharan African countries, including Sudan. Objective: We investigated the involvement of HHV-6 in paediatric central nervous system (CNS) infections in Khartoum, Sudan. Methods: Febrile patients aged up to 15 years with suspected CNS infections at Omdurman Hospital for Children from 01 December 2009 to 01 August 2010 were included. Viral DNA was extracted from leftover cerebrospinal fluid (CSF) specimens and quantitatively amplified by real-time polymerase chain reaction (PCR) at Umeå University in Sweden. Results: Of 503 CSF specimens, 13 (2.6%) were positive for HHV-6 (33.0% [13/40 of cases with proven infectious meningitis]). The median thermal cycle threshold for all HHV-6-positive specimens was 38 (range: 31.9-40.8). The median number of virus copies was 281.3/PCR run (1 × 105 copies/mL CSF; range: 30-44 × 103 copies/PCR run [12 × 103 - 18 × 106 copies/mL CSF]). All positive patients presented with fever and vomiting; 86.0% had seizures. The male-to-female ratio was 1:1; 50.0% were toddlers, 42.0% infants and 8.0% teenagers. Most (83.0%) were admitted in the dry season and 17.0% in the rainy season. Cerebrospinal fluid leukocytosis was seen in 33.0%, CSF glucose levels were normal in 86.0% and low in 14.0%, and CSF protein levels were low in 14.0% and high in 43.0%. Conclusion: Among children in Sudan with CNS infections, HHV-6 is common. Studies on the existence and spread of HHV-6 chromosomal integration in this population are needed.

First record of West Nile Virus detection inside wild mosquitoes in Khartoum capital of Sudan using PCR.

This study aimed to explore the presence of West Nile Virus (WNV) inside four species of mosquitoes: Culex univittatus (Theobald), Culex quinquefasciatus (Say) Aedes vittatus (Bigot) and Aedes vexans (Meigen). Adult wild mosquitoes were collected from different sites: Soba West, Hellat Kuku, Shambat, and Khartoum North Central Live Stock Market (KCLM). Surveys were carried out at Khartoum State during two phases: pre to the rainy season and post to the rainy season. Mosquito specimens were identified using classical keys then preserved at -80 °C freezer for two weeks till the virus examination using polymerase chain reaction (PCR) were carried out. WNV has been detected inside the three species of mosquitoes: A. vexans, C. univittatus, and C. quinquefasciatus. The species were collected from Hellat Kuku, (Shambat and Hellat Kuku), and (Shambat and KCLM) respectively. Two species of mosquitoes were positive for the virus: C. quinquefasciatus and C. univittatus. Positive results for the virus during the first phase of the study; males of C. quinquefasciatus and C. univittatus collected during the second phase of the study were also tested for the existence of the virus and they were positive. For our knowledge this study represents first record of WNV inside wild mosquitoes in Sudan. PCR technique provided reliable information because specific primer-probe sets were used for the detection of the virus. Extra studies are required to incriminate these species of mosquitoes as potential vectors of WNV.

Seewis hantavirus in common shrew (Sorex araneus) in Sweden.

BACKGROUND: Rodent borne hantaviruses are emerging viruses infecting humans through inhalation. They cause hemorrhagic fever with renal syndrome and hemorrhagic cardiopulmonary syndrome. Recently, hantaviruses have been detected in other small mammals such as Soricomorpha (shrews, moles) and Chiroptera (bats), suggested as reservoirs for potential pandemic viruses and to play a role in the evolution of hantaviruses. It is important to study the global virome in different reservoirs, therefore our aim was to investigate whether shrews in Sweden carried any hantaviruses. Moreover, to accurately determine the host species, we developed a molecular method for identification of shrews. METHOD: Shrews (n = 198), caught during 1998 in Sweden, were screened with a pan-hantavirus PCR using primers from a conserved region of the large genome segment. In addition to morphological typing of shrews, we developed a molecular based typing method using sequencing of the mitochondrial cytochrome C oxidase I (COI) and cytochrome B (CytB) genes. PCR amplified hantavirus and shrew fragments were sequenced and phylogenetically analysed. RESULTS: Hantavirus RNA was detected in three shrews. Sequencing identified the virus as Seewis hantavirus (SWSV), most closely related to previous isolates from Finland and Russia. All three SWSV sequences were retrieved from common shrews (Sorex araneus) sampled in Västerbotten County, Sweden. The genetic assay for shrew identification was able to identify native Swedish shrew species, and the genetic typing of the Swedish common shrews revealed that they were most similar to common shrews from Russia. CONCLUSION: We detected SWSV RNA in Swedish common shrew samples and developed a genetic assay for shrew identification based on the COI and CytB genes. This was the first report of presence of hantavirus in Swedish shrews.

Common Co-morbidities Are Challenging in the Diagnosis of Middle East Respiratory Syndrome (MERS-CoV) in Saudi Arabia.

BACKGROUND AND OBJECTIVE: Middle East respiratory syndrome coronavirus (MERS-CoV) is a relatively recent human disease reported initially in Saudi Arabia in September, 2012. Morbidities investigation includes a process of excluding other possible options until certain suspected cases are confirmed. MATERIAL AND METHODS: In this study, we formulated a model under the authorization of the Saudi Ministry of Health to accurately identify cases among admitted suspected cases depending on specific signs and symptoms. Real-time polymerase chain reaction was used for confirmation of the positive cases. RESULTS: The results showed that the number of patients with combined symptoms of fever/sore throat and fever/cough/SOB was significantly higher in confirmed cases than in non-confirmed cases (p<0.05). Besides, the number of confirmed MERS-CoV cases was significantly higher among cases included in the study than excluded cases. It was also clearly demonstrated that fever combined with other symptoms represents 60% of the confirmed cases, which is significantly higher than for cases with other combined symptoms (p<0.0001). CONCLUSION: To the best of our knowledge, there are no appropriate diagnostic models that can differentiate human MERS-CoV infection among other respiratory infections. Therefore, we recommend the adoption of this newly established model of MERS-CoV to short- list corona suspected cases in Saudi Arabia.

Prevalence and identification of arthropod-transmitted viruses in Kassala state, Eastern Sudan.

Vector-borne diseases are responsible for more than 20% of the infectious diseases worldwide. The prevalence of arboviruses transmit diseases to humans in Sudan has not been investigated. Mosquito-borne viral diseases increase globally incidence, including the Sudan. Frequent unknown fever outbreaks have been reported in eastern region, Sudan. However, diagnosis was based exclusively on clinical signs and symptoms without confirmatory laboratory investigations. However, for accurate detection of these viruses in outbreaks, molecular technique is considered. The objective of this study was to determine the prevalence of six arboviruses in the Kassala state of east Sudan during unknown fever outbreak. A cross sectional hospital-based study was conducted in the Kassala, Teaching Hospital. Blood samples from 119 patients suffering from unknown fever were used for screening of six arboviruses, hepatitis E virus and malarial using molecular techniques and serology.  The overall arboviruses seroprevelance was 61.3% (73/119). The highest positivity rate was 73.1% (52/73) chikungunya virus; 29 males and 20 females patients were chikungunya positive. Other arboviruses were circulating in low rate 20.5% (15/73), and 6.8% (5/73) for sindbis and rift valley fever viruses respectively. Hepatitis E virus was negative in all cases and malaria positivity rate 13.4% (16/119). The prevalence of arboviruses among unknown fever patients present to Kassala teaching hospital of eastern region in Sudan is significantly high (61.3%). The chikungunya virus is the predominant causative agent of arboviruses. Molecular techniques such as PCR are important for accurate and rapid diagnosis of this viral outbreak.

Alkhurma Hemorrhagic Fever Virus RNA in Hyalomma rufipes Ticks Infesting Migratory Birds, Europe and Asia Minor.

Alkhurma hemorrhagic fever virus RNA was detected in immature Hyalomma rufipes ticks infesting northward migratory birds caught in the North Mediterranean Basin. This finding suggests a role for birds in the ecology of the Alkhurma hemorrhagic fever virus and a potential mechanism for dissemination to novel regions. Increased surveillance is warranted.

Publisher Correction: A molecular cross-linking approach for hybrid metal oxides.

In the version of this Article originally published, Liban M. A. Saleh was incorrectly listed as Liban A. M. Saleh due to a technical error. This has now been amended in all online versions of the Article.

A molecular cross-linking approach for hybrid metal oxides.

There is significant interest in the development of methods to create hybrid materials that transform capabilities, in particular for Earth-abundant metal oxides, such as TiO2, to give improved or new properties relevant to a broad spectrum of applications. Here we introduce an approach we refer to as 'molecular cross-linking', whereby a hybrid molecular boron oxide material is formed from polyhedral boron-cluster precursors of the type [B12(OH)12]2-. This new approach is enabled by the inherent robustness of the boron-cluster molecular building block, which is compatible with the harsh thermal and oxidizing conditions that are necessary for the synthesis of many metal oxides. In this work, using a battery of experimental techniques and materials simulation, we show how this material can be interfaced successfully with TiO2 and other metal oxides to give boron-rich hybrid materials with intriguing photophysical and electrochemical properties.

Association of Rift Valley fever virus infection with miscarriage in Sudanese women: a cross-sectional study.

BACKGROUND: Rift Valley fever virus is an emerging mosquito-borne virus that causes infections in animals and human beings in Africa and the Arabian Peninsula. Outbreaks of Rift Valley fever lead to mass abortions in livestock, but such abortions have not been identified in human beings. Our aim was to investigate the cause of miscarriages in febrile pregnant women in an area endemic for Rift Valley fever. METHODS: Pregnant women with fever of unknown origin who attended the governmental hospital of Port Sudan, Sudan, between June 30, 2011, and Nov 17, 2012, were sampled at admission and included in this cross-sectional study. Medical records were retrieved and haematological tests were done on patient samples. Presence of viral RNA as well as antibodies against a variety of viruses were analysed. Any association of viral infections, symptoms, and laboratory parameters to pregnancy outcome was investigated using Pearson's χ2 test. FINDINGS: Of 130 pregnant women with febrile disease, 28 were infected with Rift Valley fever virus and 31 with chikungunya virus, with typical clinical and laboratory findings for the infection in question. 15 (54%) of 28 women with an acute Rift Valley fever virus infection had miscarriages compared with 12 (12%) of 102 women negative for Rift Valley fever virus (p<0·0001). In a multiple logistic regression analysis, adjusting for age, haemorrhagic disease, and chikungunya virus infection, an acute Rift Valley fever virus infection was an independent predictor of having a miscarriage (odds ratio 7·4, 95% CI 2·7-20·1; p<0·0001). INTERPRETATION: This study is the first to show an association between infection with Rift Valley fever virus and miscarriage in pregnant women. Further studies are warranted to investigate the possible mechanisms. Our findings have implications for implementation of preventive measures, and evidence-based information to the public in endemic countries should be strongly recommended during Rift Valley fever outbreaks. FUNDING: Schlumberger Faculty for the Future, CRDF Global (31141), the Swedish International Development Cooperation Agency, the County Council of Västerbotten, and the Faculty of Medicine, Umeå University.

Development and evaluation of a broad reacting SYBR-green based quantitative real-time PCR for the detection of different hantaviruses.

BACKGROUND: Hantaviruses are endemic in most parts of the world and cause hundreds of thousand human cases of hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) annually throughout Eurasia and the Americas. They are zoonotic viruses, most commonly transmitted to humans by aerosolized rodent excreta. New hantaviruses are frequently discovered in previously unknown reservoir species and geographic areas. Consequently, there is a need to improve hantavirus diagnostics. OBJECTIVES: This paper describes the design and evaluation of a rapid and robust quantitative real-time PCR (QRT-PCR) assay able to detect a wide range of hantaviruses. STUDY DESIGN: Primers with the potential to detect different hantaviruses were designed from conserved regions of different hantavirus L segments, as identified from multiple sequence alignments. RESULTS: By using SYBR-green-based QRT-PCR 100-1000 target molecules of in vitro produced RNA and less than 100 copies of hantavirus RNA from different hantavirus clades and regions of the world were detected. When using the assay on clinical samples from patients with acute HFRS, Puumala hantavirus (PUUV) RNA was confirmed in all previously positive samples. Notably, the broad reacting L-segment QRT-PCR also detected viral RNA in HFRS patient samples, previously negative by a QRT-PCR targeting the S segment of PUUV. CONCLUSIONS: This novel assay provides a powerful tool for diagnosis of hantaviruses from different clades and regions and may also be useful in surveys with the purpose of finding new hantaviruses in rodent or insectivore species.

Longitudinal analysis of the human T cell response during acute hantavirus infection.

Longitudinal studies of T cell immune responses during viral infections in humans are essential for our understanding of how effector T cell responses develop, clear infection, and provide long-lasting immunity. Here, following an outbreak of a Puumala hantavirus infection in the human population, we longitudinally analyzed the primary CD8 T cell response in infected individuals from the first onset of clinical symptoms until viral clearance. A vigorous CD8 T cell response was observed early following the onset of clinical symptoms, determined by the presence of high numbers of Ki67(+)CD38(+)HLA-DR(+) effector CD8 T cells. This response encompassed up to 50% of total blood CD8 T cells, and it subsequently contracted in parallel with a decrease in viral load. Expression levels of perforin and granzyme B were high throughout the initial T cell response and likewise normalized following viral clearance. When monitoring regulatory components, no induction of regulatory CD4 or CD8 T cells was observed in the patients during the infection. However, CD8 as well as CD4 T cells exhibited a distinct expression profile of inhibitory PD-1 and CTLA-4 molecules. The present results provide insight into the development of the T cell response in humans, from the very onset of clinical symptoms following a viral infection to resolution of the disease.

Broadly targeted triplex real-time PCR detection of influenza A, B and C viruses based on the nucleoprotein gene and a novel "MegaBeacon" probe strategy.

A PCR assay that covers animal and human influenza A, B and C viruses, i.e., most of Orthomyxoviridae, is needed. Influenza types are distinguished based on differences in the nucleoprotein (NP) present in the virus. Conserved NP regions were therefore used to design a TaqMan-based triplex reverse transcription real-time PCR method. Variability of influenza A within the probe target region mandated the development of a novel molecular beacon, the "Mega" molecular beacon (MegaBeacon; MegB), for the detection of influenza A with this method. MegaBeacon is a mismatch-tolerant molecular beacon that is also a TaqMan probe. The triplex method (3QPCR-MegB) was evaluated with influenza A isolates covering 18 HxNx combinations, two influenza B isolates, and five Japanese influenza C isolates, as well as influenza A, B and C synthetic DNA targets. One to ten viral RNA and cDNA genome equivalents were detected per PCR reaction for influenza A, B and C. Seventy-one human nasopharyngeal aspirates from respiratory infections yielded 30 influenza A, 11 influenza B and 0 influenza C with 3QPCR-MegB, where immunofluorescence (IF) found 28 influenza A and 10 influenza B. 3QPCR-MegB was more mismatch-tolerant than a variant PCR with an influenza A TaqMan probe (3QPCR) and is a sensitive and rational method to detect influenza viruses of animal and human origin. MegaBeacon probes hold promise for variable target nucleic acids.

Broadly targeted multiprobe QPCR for detection of coronaviruses: Coronavirus is common among mallard ducks (Anas platyrhynchos).

Coronaviruses (CoVs) can cause trivial or fatal disease in humans and in animals. Detection methods for a wide range of CoVs are needed, to understand viral evolution, host range, transmission and maintenance in reservoirs. A new concept, "Multiprobe QPCR", which uses a balanced mixture of competing discrete non- or moderately degenerated nuclease degradable (TaqMan) probes was employed. It provides a broadly targeted and rational single tube real-time reverse transcription PCR ("NQPCR") for the generic detection and discovery of CoV. Degenerate primers, previously published, and the new probes, were from a conserved stretch of open reading frame 1b, encoding the replicase. This multiprobe design reduced the degree of probe degeneration, which otherwise decreases the sensitivity, and allowed a preliminary classification of the amplified sequence directly from the QPCR trace. The split probe strategy allowed detection of down to 10 viral nucleic acid equivalents of CoV from all known CoV groups. Evaluation was with reference CoV strains, synthetic targets, human respiratory samples and avian fecal samples. Infectious-Bronchitis-Virus (IBV)-related variants were found in 7 of 35 sample pools, from 100 wild mallards (Anas platyrhynchos). Ducks may spread and harbour CoVs. NQPCR can detect a wide range of CoVs, as illustrated for humans and ducks.

Virus induces metal-binding proteins and changed trace element balance in the brain during the course of a common human infection (coxsackievirus B3) in mice.

Autopsy of the brain has shown a change in trace element balance in some virus-infected individuals, but it is not known whether this event was a result of the infection. In the present study coxsackievirus B3 (CVB3) adapted to Balb/c mice was used to study whether infection induces gene expression of the metal-binding/transporting proteins metallothionein (MT1 and MT3) and divalent-metal transporter 1 (DMT1) and whether it changes the balance of trace elements in the brain. Virus and MT1, MT3, and DMT1 were quantitatively measured by RT-PCR on days 3, 6 and 9 of the infection. Trace elements (13) were measured in serum and the brain by ICP-MS. High numbers of virus were found in the brain on days 3 and 6, but virus counts were decreased and present only in 50% of the mice on day 9. Gene expression of MT1 tended to increase on all days, whereas that of MT3 only showed a minor and not significant increase on day 3. No clear effect was observed in the expression of DMT1. The increase of MT3 was correlated to the brain concentration of Cu. The Cu/Zn ratio in serum increased as a response to the infection. There was a similar decrease in Cd in serum and the brain. On day 6 of the infection, Hg increased in the brain (p<0.05) and was positively correlated to a concomitant decrease (p<0.05) in serum. Virus numbers in the brain were on day 6 positively correlated (p<0.05) to As concentrations. Enteroviral infections may therefore be an underlying factor regarding the changes in essential as well as potentially toxic trace elements in the brain.

Experience from the development of a diagnostic single tube real-time PCR for human caliciviruses, Norovirus genogroups I and II.

Detection of caliciviruses requires high mutation tolerance and throughput. The development of a rational simple, single tube reverse transcription-real-time quantitative PCR (QPCR) technique for human noroviruses (NV) is reported here. A dual-probe, triple-primer system (NM system) was used for simultaneous detection and preliminary differentiation of NV genogroups in fecal samples. The design was based on a comprehensive analysis of all 1140 NV sequences available in GenBank. A touch-down amplification protocol improved the frequency of detection. The final QPCR was evaluated with 71 fecal samples from outbreak and sporadic cases in Sweden (1997-2004), all calicivirus-positive by electron microscopy. Up to 56 (79 %) were positive. The method is more rational than NV detection methods described previously, and should be a developmental basis for large-scale routine methods for detection of NV.

Exposure to hepatitis C virus induces cellular immune responses without detectable viremia or seroconversion.

Sporadic cases of cell-mediated immunity (CMI) in persons exposed to hepatitis C (HCV) but evidently uninfected have been reported. To further define this, we measured CMI in individuals without evidence of HCV infection, that is, negative for HCV-antibodies (anti-HCV) and RNA, residing in a rural Egyptian community where prevalence of anti-HCV was 24%. Cell-mediated immunity (CMI) measured by interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay, confirmed by intracellular staining using flow cytometry, against HCV peptides was measured in seronegative individuals with high-risk (HR) and low-risk (LR) exposures to HCV. Thirteen of 71 (18.3%) HR subjects but only 1 of 35 (2.9%) LR subjects had detectable CMI (P = 0.032). These data are compatible with the hypothesis that exposures to HCV may lead to development of HCV-specific CMI without anti-HCV and ongoing viral replication. We speculate induced CMI clears HCV sometimes when anti-HCV is not detectable, and HCV-specific CMI is a useful surrogate marker for exposure to HCV.

Quantitative PCR-enhanced immunoassay for measurement of enteroviral immunoglobulin M antibody and diagnosis of aseptic meningitis.

A PCR-enhanced immunoassay (PIA) to detect enterovirus (EV) immunoglobulin M (IgM) for diagnosis of recent EV infection was recently developed. This test was compared with another EV IgM capture technique, the solid-phase reverse immunosorbent test (SPRIST). Fourteen of 43 serum samples from aseptic meningitis patients were positive by PIA, whereas 10 were positive by SPRIST. One of 39 control serum samples was weakly positive by PIA. A single-serum-dilution real-time PCR-based PIA for EV IgM (quantitative PIA [QPIA]) was also developed and evaluated against PIA, SPRIST, an EV IgM radioimmunoassay (RIA), and clinical data. A mixture of 12 EVs was used as the antigen. Results from investigating four groups of serum samples were as follows. (i) The nine PIA-positive serum samples in group 1 were all positive by QPIA. (ii) Group 2 consisted of 59 serum samples from aseptic meningitis patients. Nineteen of 30 serum samples (63%) taken at hospital admission were positive by QPIA. Of these, 17 were positive in EV PCR. (iii) None of the 30 control serum samples in group 3 were positive by QPIA. (iv) For the 24 serum samples in group 4, of which 11 were positive and 13 were negative by RIA, the QPIA results were completely concordant. The sensitivity and specificity of QPIA for diagnosis of EV infection were 70 and 80%, respectively. QPIA provides a rational strategy for the detection of EV IgM, allows the use of viral antigens with minimal purification, and needs no virus-specific reagents apart from those in the PCR. QPIA is a generally applicable method for the detection of viral IgM in IgM capture assays.

A sensitive and quantitative single-tube real-time reverse transcriptase-PCR for detection of enteroviral RNA.

BACKGROUND: Enteroviruses (EVs) are significant human pathogens. Rapid and sensitive diagnostic techniques are desirable. OBJECTIVES: To develop a quantitative single-tube real-time reverse transcription-polymerase chain reaction (RT-PCR) for human enterovirus ribonucleic acid (RNA) (QPCR), with protection against amplimer contamination. STUDY DESIGN: The method was evaluated with serial dilutions of EV, 62 cerebrospinal fluid (CSF) specimens from meningitis patients, and the third and fourth European Union Concerted Action Enterovirus Proficiency Panels. A commercial EV PCR test was run in parallel. RESULTS: Optimisation included RNA extraction procedure, design and concentrations of primers and probes from the 5' non-coding region as well as recombinant Thermus thermophilus polymerase (rTth), Mn(OAc)(2) and thermolabile UNG concentrations. Of 62 CSF samples from cases of meningitis submitted for QPCR testing, 34 (76%) and 21 (47%) were positive by QPCR and a commercial EV RNA detection kit, respectively. The detection limit of QPCR was 0.001 TCID(50)/ml (50% tissue culture-infective dose per millilitre) for a coxsackievirus B2 preparation and <10 copies of a plasmid containing coxsackievirus B2 complementary deoxyribonucleic acid (cDNA). The relation between threshold cycle (C(t)) and amount of virus was linear (r = 0.99) over a range of 10(-3) to 10(4) TCID(50)/ml of coxsackievirus B2. CONCLUSIONS: The QPCR method allows a large number of samples to be screened rapidly. Its sensitivity, simplicity, and reproducibility make it a suitable tool for the routine laboratory.