Infected Human Blood Research Articles

An Update on Ebola Virus Epidemiology and Experimental Modalities

Ebola virus disease (EVD), formerly known as Ebola hemorrhagic fever, is a re-emerging zoonotic disease that has caused periodic outbreaks since the first reported outbreak in 1976. In 2014, in West Africa, the largest outbreak of Ebola virus in history emerged, affecting close to 30,000 people; more than 10,000 of these individuals died. This virus, which causes fatal hemorrhagic fever in humans and non-human primates, has the highest mortality rate (25-90%) among the viral hemorrhagic fever diseases. Ebola virus is transmitted through the blood and body fluids of infected animals and humans, as well as through contaminated materials. The virus is often spread in the hospital setting, and it is classified as a biosafety level 4 agent. Accordingly, diagnostic procedures and infected patient care should be performed at the highest safety levels. The potential therapies and vaccines against EVD have continued developing or been used in emergency situations during last outbreak. Some of these include the chimpanzee adenovirus type-3 and recombinant vesicular stomatitis virus-Ebola vaccines and new treatment modalities, such as passive immunization, brincidofovir, and ZMapp. Although these trials have shown different levels of success, there are no standardized treatments or vaccines that have been approved for humans. Such prevention is the only way to reduce human infection and the spread of outbreaks and is primarily based on personal protection measures and raising awareness of the risk factors.

Human Cytomegalovirus Promotes Survival of Infected Monocytes via a Distinct Temporal Regulation of Cellular Bcl-2 Family Proteins.

Monocytes play a key role in the hematogenous dissemination of human cytomegalovirus (HCMV) to target organ systems. To infect monocytes and reprogram them to deliver infectious virus, HCMV must overcome biological obstacles, including the short life span of monocytes and their antiviral proapoptotic response to infection. We have shown that virally induced upregulation of cellular Mcl-1 promotes early survival of HCMV-infected monocytes, allowing cells to overcome an early apoptotic checkpoint at around 48 h postinfection (hpi). Here, we demonstrate an HCMV-dependent shift from Mcl-1 as the primary antiapoptotic player to the related protein, Bcl-2, later during infection. Bcl-2 was upregulated in HCMV-infected monocytes beginning at 48 hpi. Treatment with the Bcl-2 antagonist ABT-199 only reduced the prosurvival effects of HCMV in target monocytes beginning at 48 hpi, suggesting that Mcl-1 controls survival prior to 48 hpi, while Bcl-2 promotes survival after 48 hpi. Although Bcl-2 was upregulated following viral binding/signaling through cellular integrins (compared to Mcl-1, which is upregulated through binding/activation of epidermal growth factor receptor [EGFR]), it functioned similarly to Mcl-1, adopting the early role of Mcl-1 in preventing caspase-3 cleavage/activation. This distinct, HCMV-induced shift from Mcl-1 to Bcl-2 occurs in response to a cellular upregulation of proapoptotic Bax, as small interfering RNA (siRNA)-mediated knockdown of Bax reduced the upregulation of Bcl-2 in infected monocytes and rescued the cells from the apoptotic effects of Bcl-2 inhibition. Our data demonstrate a distinct survival strategy whereby HCMV induces a biphasic regulation of cellular Bcl-2 proteins to promote host cell survival, leading to viral dissemination and the establishment of persistent HCMV infection. Hematogenous dissemination of HCMV via infected monocytes is a crucial component of the viral survival strategy and is required for the establishment of persistent infection and for viral spread to additional hosts. Our system of infected primary human blood monocytes provides us with an opportunity to answer specific questions about viral spread and persistence in in vivo-relevant myeloid cells that cannot be addressed with the more traditionally used replication-permissive cells. Our goal in examining the mechanisms whereby HCMV reprograms infected monocytes to promote viral dissemination is to uncover new targets for therapeutic intervention that would disrupt key viral survival and persistence strategies. Because of this important role in maintaining survival of HCMV-infected monocytes, our new data on the role of Bcl-2 regulation during viral infection represents a promising molecular target for mitigating viral spread and persistence.

Human TLR8 senses UR/URR motifs in bacterial and mitochondrial RNA.

Toll-like receptor (TLR) 13 and TLR2 are the major sensors of Gram-positive bacteria in mice. TLR13 recognizes Sa19, a specific 23S ribosomal (r) RNA-derived fragment and bacterial modification of Sa19 ablates binding to TLR13, and to antibiotics such as erythromycin. Similarly, RNase A-treated Staphylococcus aureus activate human peripheral blood mononuclear cells (PBMCs) only via TLR2, implying single-stranded (ss) RNA as major stimulant. Here, we identify human TLR8 as functional TLR13 equivalent that promiscuously senses ssRNA. Accordingly, Sa19 and mitochondrial (mt) 16S rRNA sequence-derived oligoribonucleotides (ORNs) stimulate PBMCs in a MyD88-dependent manner. These ORNs, as well as S. aureus-, Escherichia coli-, and mt-RNA, also activate differentiated human monocytoid THP-1 cells, provided they express TLR8. Moreover, Unc93b1(-/-)- and Tlr8(-/-)-THP-1 cells are refractory, while endogenous and ectopically expressed TLR8 confers responsiveness in a UR/URR RNA ligand consensus motif-dependent manner. If TLR8 function is inhibited by suppression of lysosomal function, antibiotic treatment efficiently blocks bacteria-driven inflammatory responses in infected human whole blood cultures. Sepsis therapy might thus benefit from interfering with TLR8 function.

In vivo cellular tropism of gorilla simian foamy virus in blood of infected humans.

Simian foamy viruses (SFV) are retroviruses that are widespread among nonhuman primates. SFV can be transmitted to humans, giving rise to a persistent infection. Only a few data are available concerning the distribution of SFV in human blood cells. Here we purified blood mononuclear cell subsets from 11 individuals infected with a Gorilla gorilla SFV strain and quantified SFV DNA levels by quantitative PCR. SFV DNA was detected in the majority of the CD8(+), CD4(+), and CD19(+) lymphocyte samples and rarely in CD14(+) monocyte and CD56(+) NK lymphocyte samples. The median (interquartile range [IQR]) SFV DNA counts were 16.0 (11.0 to 49.8), 11.3 (5.9 to 28.3), and 17.2 (2.0 to 25.2) copies/10(5) cells in CD8(+) T lymphocytes, CD4(+) T lymphocytes, and CD19(+) B lymphocytes, respectively. In the CD4 compartment, SFV DNA was detected in both memory and naive CD4(+) T lymphocytes. SFV DNA levels in CD4(+) T cells were positively correlated with the duration of the infection. Our study shows with a quantitative method that CD8(+), CD4(+), and B lymphocytes are major cellular targets of SFV in the blood of infected humans. Investigation of SFV infections in humans is important due to the origin of human immunodeficiency viruses (HIV) and human T cell lymphotropic viruses (HTLV) from cross-species transmission of their simian counterparts to humans. Surprisingly little is known about many aspects of the biology of SFV in infected humans, including quantitative data concerning the cellular targets of SFV in vivo. Here we show that the distribution of SFV DNA among the different leukocyte populations is not homogeneous and that viral load in CD4(+) T lymphocytes is correlated with the duration of infection. These new data will help in understanding the biology of retroviral infections in humans and can be useful in the growing field of SFV-based gene therapy.

Infection of human monocytes by Chlamydia pneumoniae and Chlamydia trachomatis: an in vitro comparative study

BackgroundAn increasing number of studies suggest that chlamydiae can infect immune cells. The altered immune cell function could contribute to the progression of several chronic inflammatory diseases.The aim of this study was to comparatively evaluate Chlamydia pneumoniae (CP) and Chlamydia trachomatis (CT) interactions with in vitro infected human blood monocytes.ResultsFresh isolated monocytes were infected with viable CP and CT elementary bodies and infectivity was evaluated by recultivating disrupted monocytes in permissive epithelial cells.The production of reactive oxygen and nitrogen species was studied in the presence of specific fluorescent probes. Moreover, TNF-α, INF-α, INF-β and INF-γ gene expression was determined.CT clearance from monocytes was complete at any time points after infection, while CP was able to survive up to 48 hours after infection. When NADPH oxydase or nitric oxide synthase inhibitors were used, CT infectivity in monocytes was restored, even if at low level, and CT recovery’s rate was comparable to CP one.CT-infected monocytes produced significantly higher levels of reactive species compared with CP-infected monocytes, at very early time points after infection. In the same meanwhile, TNF-α and INF-γ gene expression was significantly increased in CT-infected monocytes.ConclusionsOur data confirm that CP, but not CT, is able to survive in infected monocytes up to 48 hours post-infection. The delay in reactive species and cytokines production by CP-infected monocytes seems to be crucial for CP survival.

The impact of α-toxin on host cell plasma membrane permeability and cytokine expression during human blood infection by CA-MRSA USA300

This investigation examines the influence of α-toxin (Hla) expression by CA-MRSA on host immune cell integrity and cytokine expression during infection of human blood. Flow cytometry analysis of human blood infected by Staphylococcus aureus PFGE type USA300 or a USA300Δhla demonstrated that Hla expression significantly increased plasma membrane permeability of human CD14(+) monocytes. The increased susceptibility of human CD14(+) monocytes to Hla toxicity paralleled the high cell-surface expression on these cell types of ADAM10. USA300 rapidly associated with PMNs and monocytes but not T cells following inoculation of human blood. Transcription analysis indicated a strong up-regulation of proinflammatory cytokine transcription following infection of human blood by USA300 and USA300Δhla. CBAs and ELISAs determined that IL-6, IL-10, TNF-α, IFN-γ, IL-1β, IL-8, and IL-4 are significantly up-regulated during the initial phases of human blood infection by USA300 relative to mock-infected blood but failed to distinguish any significant differences in secreted cytokine protein concentrations during infection by USA300Δhla relative to USA300. Collectively, these findings demonstrate that expression of Hla by USA300 has a significant impact on human CD14(+) monocyte plasma membrane integrity but is not exclusively responsible for the proinflammatory cytokine profile induced by USA300 during the initial stages of human blood infection.

Pharmaceutical Bioprocessing: a new voice for the biopharmaceuticals community

The aim of the present work was to examine the immunopharmacological (anti-microbial and anti-inflammatory) effect of saponin on human peripheral blood mononuclear cells (PBMC) that are isolated as well as purified from infected (virally) and non-infected (normal) human whole blood samples. However, there is little information available concerning the effect of crude saponin on human PBMC in case of infected and non-infected human PBMCs. Saponin, one of the members of secondary metabolites that are isolated from aqueous leaves extract of Terminalia arjuna and Prosopis spicigera. Our group evaluated the immunopharmacological activity of saponin (0.5–30 mg/ml, 100 μl) in human PBMC were determined and measured cell proliferation using Concanvalin (Con A, 2.5 μg/ml, 50 μl) and estimate Th1 (IFN-gamma and TNF alpha) type of cytokines from cell culture supernatant including nitric oxide production. The results showed that crude saponin from Terminalia arjuna and Prosopis spicigera showed its inhibitory activity at higher doses (30 mg/ml, 100 μl) in Con A induced PBMC (infected and non-infected) proliferation. In addition, these saponin also inhibits its Th1 (IFN-gamma and TNF alpha) type of cytokines from Con A stimulated cell culture supernatant of PBMC and also inhibits nitric oxide production in a dose dependent manner. In conclusion, saponin appears to have potential as an anti-microbial and anti-inflammatory agent.

A pathogenic picornavirus acquires an envelope by hijacking cellular membranes

Animal viruses are broadly categorized structurally by the presence or absence of an envelope composed of a lipid-bilayer membrane1, attributes that profoundly affect stability, transmission, and immune recognition. Among those lacking an envelope, the Picornaviridae are a large and diverse family of positive-strand RNA viruses that includes hepatitis A virus (HAV), an ancient human pathogen that remains a common cause of enterically-transmitted hepatitis2–4. HAV infects in a stealth-like manner and replicates efficiently in the liver5. Virus-specific antibodies appear only after 3–4 weeks of infection, and typically herald its resolution3,4. Although unexplained mechanistically, both anti-HAV antibody and inactivated whole-virus vaccines prevent disease when administered as late as 2 weeks after exposure6, when virus replication is well established in the liver5. Here, we show that HAV released from cells is cloaked in host-derived membranes, thereby protecting the virion from antibody-mediated neutralization. These enveloped viruses (“eHAV”) resemble exosomes7, small vesicles that are increasingly recognized to play important roles in intercellular communications. They are fully infectious, sensitive to chloroform extraction, and circulate in the blood of infected humans. Their biogenesis is dependent upon host proteins associated with endosomal-sorting complexes required for transport (ESCRT)8, VPS4B and ALIX. While the hijacking of membranes by HAV facilitates escape from neutralizing antibodies and likely promotes virus spread within the liver, anti-capsid antibodies restrict replication following infection with eHAV, suggesting a possible explanation for post-exposure prophylaxis. Membrane hijacking by HAV blurs the classic distinction between “enveloped” and “nonenveloped” viruses, and has broad implications for mechanisms of viral egress from infected cells as well as host immune responses.

First International External Quality Assessment of Molecular Detection of Crimean-Congo Hemorrhagic Fever Virus

Crimean-Congo hemorrhagic fever (CCHF) is a zoonosis caused by a Nairovirus of the family Bunyaviridae. Infection is transmitted to humans mostly by Hyalomma ticks and also by direct contact with the blood or tissues of infected humans or viremic livestock. Clinical features usually include a rapid progression characterized by hemorrhage, myalgia and fever, with a lethality rate up to 30%. CCHF is one of the most widely distributed viral hemorrhagic fevers and has been reported in Africa, the Middle East and Asia, as well as parts of Europe. There is no approved vaccine or specific treatment against CCHF virus (CCHFV) infections. In this context, an accurate diagnosis as well as a reliable surveillance of CCHFV infections is essential. Diagnostic techniques include virus culture, serology and molecular methods, which are now increasingly used. The European Network for the Diagnostics of “Imported” Viral Diseases organized the first international external quality assessment of CCHVF molecular diagnostics in 2011 to assess the efficiency and accurateness of CCHFV molecular methods applied by expert laboratories. A proficiency test panel of 15 samples was distributed to the participants including 10 different CCHFV preparations generated from infected cell cultures, a preparation of plasmid cloned with the nucleoprotein of CCHFV, two CCHFV RNA preparations and two negative controls. Forty-four laboratories worldwide participated in the EQA study and 53 data sets were received. Twenty data sets (38%) met all criteria with optimal performance, 10 (19%) with acceptable performance, while 23 (43%) reported results showing a need for improvement. Differences in performance depended on the method used, the type of strain tested, the concentration of the sample tested and the laboratory performing the test. These results indicate that there is still a need for improving testing conditions and standardizing protocols for the molecular detection of Crimean-Congo hemorrhagic fever virus.

Shutoff of BZLF1 Gene Expression Is Necessary for Immortalization of Primary B Cells by Epstein-Barr Virus

The BZLF1 gene controls the switch between latent and lytic infection by Epstein-Barr virus (EBV). We previously reported that both the ZV and ZIIR elements within the BZLF1 promoter, Zp, are potent transcription silencers within the context of an intact EBV genome. We report here identification of another sequence element, ZV', which synergized with ZV in repressing Zp via binding ZEB1 or ZEB2. We then determined the phenotype of a variant of EBV strain B95.8 in which the ZV, ZV', and ZIIR elements were concurrently mutated. HEK293 cell lines infected with this triple mutant (tmt) virus spontaneously synthesized 6- to 10-fold more viral BZLF1, BRLF1, BMRF1, and BLLF1 RNAs, 3- to 6-fold more viral Zta, Rta, and EAD proteins, 3- to 5-fold more viral DNA, and 7- to 9-fold more infectious virus than did 293 cell lines latently infected with either the ZV ZV' double mutant (dmt) or ZIIR mutant (mt) virus. While ZV ZV' ZIIR tmt EBV efficiently infected human primary blood B cells in vitro, it was highly defective in immortalizing them. Instead of the nearly complete silencing of BZLF1 gene expression that occurs within 4 days after primary infection with wild-type EBV, the ZV ZV' ZIIR tmt-infected cells continued to synthesize BZLF1 RNA, with 90% of them dying within 9 days postinfection. BL41 cells infected with this "superlytic" virus also exhibited increased synthesis of BZLF1 and BMRF1 RNAs. Thus, we conclude that the ZV, ZV', and ZIIR silencing elements act synergistically to repress transcription from Zp, thereby tightly controlling BZLF1 gene expression, which is crucial for establishing and maintaining EBV latency.

Functional Characterization of a SUMO Deconjugating Protease of Plasmodium falciparum Using Newly Identified Small Molecule Inhibitors

Small ubiquitin-related modifier (SUMO) is implicated in the regulation of numerous biological processes including transcription, protein localization, and cell cycle control. Protein modification by SUMO is found in Plasmodium falciparum; however, its role in the regulation of the parasite life cycle is poorly understood. Here we describe functional studies of a SUMO-specific protease (SENP) of P.falciparum, PfSENP1 (PFL1635w). Expression of the catalytic domain of PfSENP1 and biochemical profiling using a positional scanning substrate library demonstrated that this protease has unique cleavage sequence preference relative to the human SENPs. In addition, we describe a class of small molecule inhibitors of this protease. The most potent lead compound inhibited both recombinant PfSENP1 activity and P.falciparum replication in infected human blood. These studies provide valuable new tools for the study of SUMOylation in P.falciparum.

Analysis of Salmonella enterica Serotype Paratyphi A Gene Expression in the Blood of Bacteremic Patients in Bangladesh

Background Salmonella enterica serotype Paratyphi A is a human-restricted cause of paratyphoid fever, accounting for up to a fifth of all cases of enteric fever in Asia.Methodology/Principal FindingsIn this work, we applied an RNA analysis method, Selective Capture of Transcribed Sequences (SCOTS), and cDNA hybridization-microarray technology to identify S. Paratyphi A transcripts expressed by bacteria in the blood of three patients in Bangladesh. In total, we detected 1,798 S. Paratyphi A mRNAs expressed in the blood of infected humans (43.9% of the ORFeome). Of these, we identified 868 in at least two patients, and 315 in all three patients. S. Paratyphi A transcripts identified in at least two patients encode proteins involved in energy metabolism, nutrient and iron acquisition, vitamin biosynthesis, stress responses, oxidative stress resistance, and pathogenesis. A number of detected transcripts are expressed from PhoP and SlyA-regulated genes associated with intra-macrophage survival, genes contained within Salmonella Pathogenicity Islands (SPIs) 1–4, 6, 10, 13, and 16, as well as RpoS-regulated genes. The largest category of identified transcripts is that of encoding proteins with unknown function. When comparing levels of bacterial mRNA using in vivo samples collected from infected patients to samples from in vitro grown organisms, we found significant differences for 347, 391, and 456 S. Paratyphi A transcripts in each of three individual patients (approximately 9.7% of the ORFeome). Of these, expression of 194 transcripts (4.7% of ORFs) was concordant in two or more patients, and 41 in all patients. Genes encoding these transcripts are contained within SPI-1, 3, 6 and 10, PhoP-regulated genes, involved in energy metabolism, nutrient acquisition, drug resistance, or uncharacterized genes. Using quantitative RT-PCR, we confirmed increased gene expression in vivo for a subset of these genes.Conclusion/SignificanceTo our knowledge, we describe the first microarray-based transcriptional analysis of a pathogen in the blood of naturally infected humans.

Current treatment of Crimean–Congo hemorrhagic fever in children

Crimean–Congo hemorrhagic fever (CCHF) is a fatal viral hemorrhagic fever and is usually transmitted to humans by tick bite, or exposure to infected blood or tissues of infected livestock or humans. Although children can be infected with the CCHF virus, infection is unusual in the younger age group. Early diagnosis and treatment of CCHF infection is critical to the survival of patients and the control of the disease. In this article, we underline current therapeutic approaches to CCHF infection in children.

Kinetic Analysis of Ex Vivo Human Blood Infection by Leishmania

The leishmanioses, vector-borne diseases caused by the trypanosomatid protozoan Leishmania, are transmitted to susceptible mammals by infected phlebotomine sand flies that inoculate promastigotes into hemorrhagic pools created in host skin. We assumed that promastigotes are delivered to a blood pool, and analyzed early promastigote interactions (0–5 min) with host components, which lead to parasite endocytosis by blood leukocytes, and to host infection. Promastigotes were incubated with NHS or with heparinized blood in near-physiological conditions, and we used cell radioimmunoassay and flow cytometry to measure the on-rate constants (k+1) of promastigote interactions with natural opsonins and erythrocytes. We obtained quantitative data for parasitized cells to determine the time-course of promastigote binding and internalization by blood leukocytes. In these reactions, promastigotes bind natural opsonins, immune adhere to erythrocytes and activate complement cytolysis, which kills ∼95% of promastigotes by 2 min post-infection. C3-promastigote binding is a key step in opsonization; nascent C3-promastigotes are the substrate for two simultaneous reactions, C3-promastigote immune adherence (IA) to erythrocytes and complement-mediated promastigote killing. The k+1 for IA was 75-fold greater than that for promastigote killing, showing that IA facilitates promastigote endocytosis and circumvents lysis. At 5 min post-infection, when reaction velocity is still linear and promastigote concentration is not limiting, 17.4% of granulocytes and 10.7% of monocytes had bound promastigotes, of which ∼50% and ∼25%, respectively, carried surface-bound (live) or internalized (live and dead) leishmanias. Of other leukocyte types, 8.5% of B cells bound but did not internalize promastigotes, and T cells, NK cells and CD209+ dendritic cells did not bind parasites. These data show that, once in contact with blood, promastigote invasion of human leukocytes is an extremely rapid and efficient reaction, and suggest that the IA reaction constitutes a central strategy for this parasite in subverting host innate immune defenses.

Helminthic Infections

Helminthic parasites are multicellular worms that possess differentiated organ systems. They (with a few exceptions) do not replicate in the human host, and they tend to elicit eosinophilia within the tissues and blood of infected humans. Helminthic parasites include nematodes (roundworms), cestodes (tapeworms), and trematodes (flukes). The major intestinal nematodes are roundworm, pinworm, hookworm, whipworm, and Strongyloides stercoralis. Trichinellosis is caused by five species of the nematode Trichinella and develops after ingestion of infected meat, usually pork or the meat of certain carnivores. Nematode infections of the major tissue are anisakiasis, visceral larva migrans,Angiostrongylus cantonensis infection, mammomonogamosis (syngamosis), gnathostomiasis, dracunculiasis, and filariasis. Trematode and cestode infections are also described in this chapter, including infections by fish, beef, and pork tapeworms, as well as cysticercosis. Disease from Paragonimus, Clonorchis, Fasciola, Fasciolopsis, and Schistosoma is covered. Echinococcus infection and hydatid cyst disease are discussed. Tables describe intestinal nematode infection and treatment and filarial parasites of humans. Figures illustrate a variety of helminthic parasites and their life cycles. This review contains 136 references.

Unified Parasite Lactate Dehydrogenase and Histidine-Rich Protein ELISA for Quantification of Plasmodium falciparum

There is a need for more objective and quantitative tools to replace microscopy in malaria diagnosis. Emphasis has recently been placed on alternative methods such as immunochromatography-based rapid tests. However, these tests provide only qualitative results. Two bio-molecules, parasite lactate dehydrogenase (pLDH) and histidine-rich proteins (HRPs), that are released by the intra-erythrocytic stages of the parasite offer certain specific characteristics that could potentially improve malaria diagnosis. In this paper, we describe a protocol for a unified sandwich ELISA that allows for the separate but concurrent measurement of pLDH and HRP biomolecules in aliquots taken from the same samples. Freshly drawn blood from a healthy unexposed adult male was used to serially dilute in vitro cultivated and synchronized ring stage Plasmodium falciparum parasites. Commercially available ELISA formats were modified to allow for the measurement of pLDH and HRP from aliquots of the same samples. The pLDH and HRP levels in the samples spiked with known numbers of infected red blood cells (iRBCs) were measured, and the values were used to generate standard graphs. The standard graphs were used to estimate the numbers of iRBCs in test samples. Serially diluted recombinant proteins were similarly used to generate a calibration curve, allowing for the expression of test results in nanograms of their respective recombinant protein. Levels of pLDH and HRPs were determined by using 1) P. falciparum culture material (cells and medium) 2) P. falciparum infected human blood (N = 6) samples, and 3) plasma from P. falciparum-infected patient (N = 22) samples. The parasite density of all culture and infected patient samples was also estimated by microscopy. Both pLDH and HRP levels correlated positively with the parasite density assessed by microscopy: Pearson correlation coefficient pLDH (r = 0.754, P < 0.0001, 95% CI: 0.47-0.89); HRP (r = 0.552, P < 0.007, 95% CI: 0.16-0.79). The HRPs seem to be released in larger quantities than pLDH (in a ratio of ~1 pLDH:~6 HRP), making the detection of HRP in culture material, blood, and plasma easier. The modified ELISA assay with quantitative measurement of pLDH and HRPs may provide a valuable tool for malaria research and patient management.

A preliminary report on Crimean-Congo haemorrhagic fever in Turkey, March - June 2008

Crimean-Congo haemorrhagic fever (CCHF) is a disease caused by a virus belonging to Bunyaviridae family. CCHF virus isolation and/or disease have been reported from more than 30 countries in Africa, Asia, south-eastern Europe, and the Middle East [1]. The main transmission routes of the virus are tick-bite and contact with tissues, body fluids and blood of infected animals [1-4]. Nosocomial transmission is another important route of infection [1]. The incubation period is generally described as 1-3 days after tick-bite and 5-6 days after exposure to infected animal or human blood or body fluid, but it can be longer. Fever, chills, headache, fatigue and myalgia are the most common symptoms in the pre-haemorrhagic period. The disease progresses to haemorrhagic form in severe cases [1]. The fatality rate of disease is reported between 7.5-50% in hospitalised patients [4-7]. This wide range may due to phylogenetic variation of the virus, transmission route and different treatment facilities [4-7].

Breastfeeding in Crimean-Congo haemorrhagic fever

Crimean-Congo haemorrhagic fever (CCHF) is potentially a fatal disease transmitted by tick bite, close contact with blood or tissues of infected humans or viraemic livestock. We present the clinical course of 2 breastfeeding women with CCHF and their babies. Both the mothers had positive reverse transcriptase-polymerase chain reaction for CCHF virus in blood and it was negative in breast milk. At follow-up, babies did not develop CCHF infection.

The presence of infectious extracellular Francisella tularensis subsp. novicida in murine plasma after pulmonary challenge

Francisella tularensis is a gram-negative, facultative intracellular bacterium and is the causative agent of the zoonotic disease tularemia [1]. F. tularensis has been considered a potential biological weapon due to its low infectious dose and high mortality rate [2]. F. tularensis can be classified into several subspecies, including those relevant to human disease: F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B); F. novicida and F. mediasiatica [3]. However, most of our knowledge about the pathogenesis of Francisella and the immune responses to the infection have come from studies of F. tularensis LVS (derived from holartica) and F. novicida [4–6]. Both organisms are attenuated in humans, while retaining virulence in mice. It is well established that Francisella enters and replicates within host cells, and a strong research focus has been on deciphering the mechanisms for intramacrophage growth. However, little is known about the extracellular phase of Francisella. F. tularensis has been cultured from blood of infected animals and humans, although the reported cases have been rare [7, 8]. Specifically, Long et al. [8] demonstrated that LVS could be recovered from plasma prepared from mice 72 h after intravenous infection. These extracellular bacteria accounted for less than 1% of total LVS within the blood. However, a recent study by Forestal et al. [9] indicated a much higher percentage (>50%) of extracelluar Francisella (type A SCHU S4 and LVS) within plasma of mice infected intradermally or intranasally. Here, independently, we confirm the presence of extracellular Francisella in plasma after intranasal F. novicida challenge in a murine model of pulmonary tularemia [6]. Moreover, we demonstrate that the extracellular Francisella in plasma is highly infectious and that this phase may contribute to the rapid dissemination of the bacterium from lungs to the liver after pulmonary infection. F. novicida strain U112 shares a high degree of antigenic and genetic similarity with F. tularensis type A and B [10] and is highly virulent in mice with a LD50 of approximately 10 colony forming units (CFU) by the intranasal (i.n.) route [6]. In this study, we monitored the replication and dissemination of F. novicida in mice. Bacteria were grown at 37°C in Trypticase Soy Broth (TSB) supplemented with 0.1% cysteine. The concentrations of the bacterial inocula were determined by serial dilution plating on Trypticase Soy Agar (TSA). Female BALB/c mice (6–8 weeks old, obtained from the Jackson Laboratory, Bar Harbor, ME) were challenged i.n. with 400 CFU (40 LD50). All animal care and experimental procedures were performed in compliance with the Institutional Animal Care and Use Committee guidelines of the University of Texas at San Antonio. Bacterial loads in the lungs and livers were assessed by plating tissue homogenates on TSA. Within 24 h of pulmonary challenge, a large number of F. novicida, approximately 1×10 CFU/g tissue, was observed in the lungs, which increased to 1×10 CFU/g tissue by 48 h and then decreased to 1×10 CFU/g tissue by 72 h. In the livers of infected mice, there was a progressive increase in bacterial load with levels rising from 100 CFU/g tissue at 24 h to approximately 1×10 CFU/g tissue by 72 h (Fig. 1a). These results demonstrate dissemination of F. novicida from the lungs to the secondary organs after pulmonary exposure. To determine a probable mechanism for the rapid spread to the secondary organs, blood was collected from the orbital plexus at 24, 48 and 72 h Eur J Clin Microbiol Infect Dis (2008) 27:323–325 DOI 10.1007/s10096-007-0434-x

MALARIA IN EXPERIMENTAL BIOLOGY

![Figure][1] The hard statistics about malaria are far from comforting. Killing over one million victims every year and infecting another 300 million, the disease continues to have a catastrophic effect in many developing countries, striking the most vulnerable members of society: pregnant