Inflammatory responses in Ebola virus-infected patients.

Ebola virus subtype Zaire (Ebo-Z) induces acute haemorrhagic fever and a 60-80% mortality rate in humans. Inflammatory responses were monitored in victims and survivors of Ebo-Z haemorrhagic fever during two recent outbreaks in Gabon. Survivors were characterized by a transient release in plasma of interleukin-1beta (IL-1beta), IL-6, tumour necrosis factor-alpha (TNFalpha), macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta early in the disease, followed by circulation of IL-1 receptor antagonist (IL-1RA) and soluble receptors for TNFalpha (sTNF-R) and IL-6 (sIL-6R) towards the end of the symptomatic phase and after recovery. Fatal infection was associated with moderate levels of TNFalpha and IL-6, and high levels of IL-10, IL-1RA and sTNF-R, in the days before death, while IL-1beta was not detected and MIP-1alpha and MIP-1beta concentrations were similar to those of endemic controls. Simultaneous massive activation of monocytes/macrophages, the main target of Ebo-Z, was suggested in fatal infection by elevated neopterin levels. Thus, presence of IL-1beta and of elevated concentrations of IL-6 in plasma during the symptomatic phase can be used as markers of non-fatal infection, while release of IL-10 and of high levels of neopterin and IL-1RA in plasma as soon as a few days after the disease onset is indicative of a fatal outcome. In conclusion, recovery from Ebo-Z infection is associated with early and well-regulated inflammatory responses, which may be crucial in controlling viral replication and inducing specific immunity. In contrast, defective inflammatory responses and massive monocyte/macrophage activation were associated with fatal outcome.

Early immune responses accompanying human asymptomatic Ebola infections.

In a recent study we identified certain asymptomatic individuals infected by Ebola virus (EBOV) who mounted specific IgG and early and strong inflammatory responses. Here, we further characterized the primary immune response to EBOV during the course of asymptomatic infection in humans. Inflammatory responses occurred in temporal association with anti-inflammatory phase composed by soluble antagonist IL-1RA, circulating TNF receptors, IL-10 and cortisol. At the end of the inflammatory process, mRNA expression of T-cell cytokines (IL-2 and IL-4) and activation markers (CD28, CD40L and CTLA4) was up-regulated, strongly suggesting T-cell activation. This T-cell activation was followed by EBOV-specific IgG responses (mainly IgG3 ang IgG1), and by marked and sustained up-regulation of IFN gamma, FasL and perforin mRNA expression, suggesting activation of cytotoxic cells. The terminal down-regulation of these latter markers coincided with the release of the apoptotic marker 41/7 NMP in blood and with the disappearance of viral RNA from PBMC, suggesting that infected cells are eliminated by cytotoxic mechanisms. Finally, RT-PCR analysis of TCR-V beta repertoire usage showed that TCR-V beta 12 mRNA was never expressed during the infection. Taken together, these findings improve our understanding about immune response during human asymptomatic Ebola infection, and throw new light on protection against Ebola virus.

Human asymptomatic Ebola infection and strong inflammatory response.

BACKGROUND: Ebola virus is one of the most virulent pathogens, killing a very high proportion of patients within 5-7 days. Two outbreaks of fulminating haemorrhagic fever occurred in northern Gabon in 1996, with a 70% case-fatality rate. During both outbreaks we identified some individuals in direct contact with sick patients who never developed symptoms. We aimed to determine whether these individuals were indeed infected with Ebola virus, and how they maintained asymptomatic status. METHODS: Blood was collected from 24 close contacts of symptomatic patients. These asymptomatic individuals were sampled 2, 3, or 4 times during a 1-month period after the first exposure to symptomatic patients. Serum samples were analysed for the presence of Ebola antigens, virus-specific IgM and IgG (by ELISA and western blot), and different cytokines and chemokines. RNA was extracted from peripheral blood mononuclear cells, and reverse transcriptase-PCR assays were done to amplify RNA of Ebola virus. PCR products were then sequenced. FINDINGS: 11 of 24 asymptomatic individuals developed both IgM and IgG responses to Ebola antigens, indicating viral infection. Western-blot analysis showed that IgG responses were directed to nucleoprotein and viral protein of 40 kDa. The glycoprotein and viral protein of 24 kDa genes showed no nucleotide differences between symptomatic and asymptomatic individuals. Asymptomatic individuals had a strong inflammatory response characterised by high circulating concentrations of cytokines and chemokines. INTERPRETATION: This study showed that asymptomatic, replicative Ebola infection can and does occur in human beings. The lack of genetic differences between symptomatic and asymptomatic individuals suggest that asymptomatic Ebola infection did not result from viral mutations. Elucidation of the factors related to the genesis of the strong inflammatory response occurring early during the infectious process in these asymptomatic individuals could increase our understanding of the disease.

Diagnosis of Ebola haemorrhagic fever by RT-PCR in an epidemic setting.

This study reports the first field evaluation of a new diagnostic technique for Ebola virus disease with sensitivity and specificity. Ebola virus causes rare but fulminating outbreaks in Equatorial Africa. Rapid differentiation from other infections is critical for timely implementation of public health measures. Patients usually die before developing antibodies, necessitating rapid virus detection. A reverse transcriptase-polymerase chain reaction (RT-PCR) assay was developed, implemented and evaluated at Centre International de Recherches Médicales de Franceville (CIRMF) in Gabon, to detect Ebola viral RNA in peripheral blood mononuclear cells (PBMC). Twenty-six laboratory-confirmed patients during and 5 after the acute phase of Ebola haemorrhagic fever, 15 healthy controls and 20 febrile patients not infected with Ebola virus were studied. RT-PCR results were compared with ELISA antigen capture, and Ebola specific IgM and IgG antibody detection. Ebola virus RNA was amplified from 26/26 specimens from the acute phase, 3/5 during recovery, 0/20 febrile patients and 1/15 negative controls. Sensitivity of RT-PCR in identifying acute infection and early convalescence compared with antigen or IgM detection was 100% and 91% respectively, and specificity compared with antigen detection and IgM assay combined was 97%. Antigen capture detected only 83% of those identified by PCR, and IgM only 67%. Ebola virus RNA was detected in all 13 fatalities, only 5 of whom had IgM and none IgG. RT-PCR detected Ebola RNA in PBMC one to three weeks after disappearance of symptoms when antigen was undetectable. RT-PCR was the most sensitive method and able to detect virus from early acute disease throughout early recovery.

Impairment of natural killer cell activity in Chlamydia trachomatis infected individuals.

Natural killer (NK) cell activity is impaired in Chlamydia trachomatis-infected patients. The mechanisms behind the altered NK functions are not clear, but data concerning NK and antibody-dependent cellular cytotoxicity (ADCC) activity have been reported. To investigate whether this impairment is related to a defect at the target cell binding and/or the postbinding level, we evaluated highly purified NK cells obtained from 125 C. trachomatis-infected patients and compared them with 101 normal controls for their ability to kill K-562 and U-937 cell lines using a 51Cr release assay; release tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma); and kill anti-IgM preincubated P-815 cell line (ADCC activity). We found a decrease in the lytic capability of NK cells from C. trachomatis-infected patients against target cell lines; decreased ability to kill bound target cells; and low levels of released TNF-alpha and INF-gamma after incubation with U-937 cells. Taken together, these findings suggest that the impaired NK cell reaction during chlamydial infection is related to defects both at the target and postbinding levels. However, the precise mechanisms remain to be determined. The inability to restore normal NK activity after long-term culture in the presence of high levels of recombinant IL-2 support the hypothesis of an anergic process during chlamydial infection.

Defective humoral responses and extensive intravascular apoptosis are associated with fatal outcome in Ebola virus-infected patients.

Ebola virus is very pathogenic in humans. It induces an acute hemorrhagic fever that leads to death in about 70% of patients. We compared the immune responses of patients who died from Ebola virus disease with those who survived during two large outbreaks in 1996 in Gabon. In survivors, early and increasing levels of IgG, directed mainly against the nucleoprotein and the 40-kDa viral protein, were followed by clearance of circulating viral antigen and activation of cytotoxic T cells, which was indicated by the upregulation of FasL, perforin, CD28 and gamma interferon mRNA in peripheral blood mononuclear cells. In contrast, fatal infection was characterized by impaired humoral responses, with absent specific IgG and barely detectable IgM. Early activation of T cells, indicated by mRNA patterns in peripheral blood mononuclear cells and considerable release of gamma interferon in plasma, was followed in the days preceding death by the disappearance of T cell-related mRNA (including CD3 and CD8). DNA fragmentation in blood leukocytes and release of 41/7 nuclear matrix protein in plasma indicated that massive intravascular apoptosis proceeded relentlessly during the last 5 days of life. Thus, events very early in Ebola virus infection determine the control of viral replication and recovery or catastrophic illness and death.

Alloreactivity and association of human natural killer cells with the major histocompatibility complex.

All NK cells potentially lytic for autologous cells but not expressing self-major histocompatibility complex (MHC)-reactive receptors could be eliminated by a negative selection mechanism during ontogeny. This idea is based on the existence of a NK cell subset expressing a specific inhibitory receptor for allogeneic MHC alleles. As ancestral haplotypes of the MHC appear to define identical MHC haplotypes in unrelated individuals, unrelated individuals having the same ancestral haplotype should also have the same NK-defined allospecificities that have been shown to map to the human MHC. To test this prediction, multiple cell lines from unrelated individuals having the same ancestral haplotypes were tested for the NK-defined allospecificities. It was found that cells having the same ancestral haplotypes do have the same NK-defined specificities. Furthermore, the NK-defined phenotype of cells that possess two different ancestral haplotypes can be predicted from the NK-defined phenotypes of unrelated cells that are homozygous for the ancestral haplotypes concerned. Although the group 1 and 2 NK-defined allospecificities can be explained to some extent by HLA-C alleles, evidence is presented that additional genes may modify the phenotype conferred by HLA-C.

Ebola hemorrhagic fever outbreaks in Gabon, 1994-1997: epidemiologic and health control issues.

From the end of 1994 to the beginning of 1995, 49 patients with hemorrhagic symptoms were hospitalized in the Makokou General Hospital in northeastern Gabon. Yellow fever (YF) virus was first diagnosed in serum by use of polymerase chain reaction followed by blotting, and a vaccination campaign was immediately instituted. The epidemic, known as the fall 1994 epidemic, ended 6 weeks later. However, some aspects of this epidemic were atypical of YF infection, so a retrospective check for other etiologic agents was undertaken. Ebola (EBO) virus was found to be present concomitantly with YF virus in the epidemic. Two other epidemics (spring and fall 1996) occurred in the same province. GP and L genes of EBO virus isolates from all three epidemics were partially sequenced, which showed a difference of <0.1% in the base pairs. Sequencing also showed that all isolates were very similar to subtype Zaire EBO virus isolates from the Democratic Republic of the Congo.

[Serological investigation of toxoplasmosis in patients of the M.I.P. center of Franceville (Gabon)]. / Enquête sérologique sur la toxoplasmose chez les consultantes du centre de P.M.I. de Franceville (Gabon).

The seroprevalence of toxoplasmosis was assessed between 1995 and 1997 on 767 pregnant women on the occasion of their medical check-ups for pregnancy in the preventive health centre of Franceville, province of the Haut-Ogooué, Gabon. Among the women under investigation, 71.2% were found to be IgG seropositive, including 2.6% IgM seropositive. When compared to similar studies conducted for the last 20 years in the same region, these results give evidence of an increase of the seroprevalence to toxoplasmosis among pregnant women, contributing to a decreased risk of contracting the disease during pregnancy.

[Ascariasis of the cervix]. / Ascaridiose du col utérin.

The authors report a case of ascaris lumbricoïdes located in the cervix uterus of a patient suffering from gonococcal salpingitis. Reviewing the literature, they conclude that the most likely way to the parts genitals was a transanal migration toward vagina.

[Role of Schistosoma mansoni bilharziasis in male hypogonadism]. / Rôle de la bilharziose à Schistosoma mansoni dans l'hypogonadisme du mâle.

In tropical and subtropical areas, schistosomiasis may cause anatomic anomalies of the genital organs responsible for permanent or reversible infertility. Furthermore, it has been suggested that this parasitic infection may have adverse consequences on the endocrine system. To specify the effects of schistosomiasis on endocrine function, production of pituitary gonadotropins and testosterone in rats and hamsters experimentally infected with Schistosoma mansoni was studied. Prior to infection, hormone levels were within the normal range for our laboratory in all animals studied. Hormone levels (FSH, LH and testosterone) fell significantly in all experimentally infected animals, as compared with uninfected controls. These data suggest that the inhibitory effect (documented by the fall in all the hormone parameters studied) of Schistosoma mansoni on gonadal function is due to an action at the hypothalamic or pituitary level. The potential role of parasite ecdysteroids in the mechanism of action of schistosomes deserves to be studied.

Ecdysteroid-like compounds in the serum and urine of African patients infected with Loa loa and Mansonella perstans microfilariae.

Ecdysteroids are compounds related to 20-hydroxyecdysone, the insect moulting hormone. Surprisingly, they have been found in serum and urine of patients infected with helminths. In these cases, the substances are assumed to be produced by the parasites and, therefore, might be used as a marker of parasitic infection. Thus, we need to know exactly which species, at which developmental stage, can release ecdysteroids in such large quantities that they could be detected in the biological fluids of the host. Large-scale investigations must, accordingly, be devoted to the major species of helminths. In the present study, we examined 100 African patients with Loa loa and/or Mansonella perstans microfilaraemia. About 70 of them had high levels of ecdysteroid-like materials in serum or urine. In contrast, uninfected patients and European controls had much lower concentrations. However, the ecdysteroid titres did not reflect the concentration of microfilariae actually present in the blood, and some heavily infected patients were even negative. Nevertheless, the most important point was that high ecdysteroid levels in man were always associated with a pathological condition. The precise significance of the phenomenon should be determined.

Hypogonadism and ecdysteroid production in Loa loa and Mansonella perstans filariasis.

Possible endocrinological repercussions of infection with Loa loa and Mansonella perstans filariae were studied in Gabonese subjects. Microfilaremic males were compared with amicrofilaremic controls. In the infected group 13/105 subjects (12%) presented only abnormally low serum levels of testosterone (less than 4 ng/ml), 25/105 (24%) only abnormally high serum levels of gonadotrophins, FSH (greater than 15 mIU/ml) and LH (greater than 20 mIU/ml), and 22/105 (21%) presented anomalies in both testosterone and gonadotrophin levels. One out of 68 control subjects had 3.6 ng/ml seric testosterone and all had normal levels of gonadotrophins. Ecdysteroids were detected (greater than 0.025 ng/ml) in the serum of 87/97 (90%) microfilaremic subjects (GM 0.123 ng/ml) compared to 12/64 (19%) controls (GM 0.030 ng/ml). Ecdysteroids were detected in the urine of all subjects, infected (GM 8.468 ng/ml) as well as control (GM 1.245 ng/ml). The hormonal perturbations were correlated with the levels of Loa loa microfilaremia but not with those of serum and urinary ecdysteroids. These results demonstrate that microfilaremic subjects often show endocrinal signs of hypogonadism and present appreciable levels of ecdysteroids in serum and urine. A direct role for parasitic ecdysteroids in hypogonadism remains to be demonstrated.

Steroid and gonadotropin hormone levels in young African women with filarial infection.

Serum levels of dehydroepiandrosterone sulfate (DHEAS), testosterone (T), progesterone (P), estradiol (E2), prolactin (PRL), cortisol (F) and gonadotropins (FSH, LH) were analysed by radioimmunoassay for 125 schoolgirls aged 14-16, in a zone of endemic filariasis 3 days after menses. Two groups were identified: the infected group in which 38 subjects had circulating Loa loa and or Mansonella perstans microfilariae as determined by the Knott's concentration technique, and the non-infected group (87 subjects without microfilaremia). All results are expressed as the mean +/- SD. No significant difference was found between the two groups for age (14.47 +/- 1.37 yr vs 14.50 +/- 1.37 yr) or for body wt (46.10 +/- 8.45 kg vs 47.06 +/- 8.26 kg). There was a tendency to lower levels of DHEAS in the infected group by comparison with controls (54.92 +/- 37.34 micrograms/dl vs 66.80 +/- 47.18 micrograms/dl) while in the same infected group more subjects had higher levels of prolactin by comparison with the control group (10.85 +/- 14.16 ng/ml vs 9.80 +/- 5.56 ng/ml). Testosterone, progesterone, estradiol levels and the LH/FSH ratio were lower in the infected group than in the non-infected group (P: 0.25 +/- 0.12 ng/ml vs 0.33 +/- 0.20 ng/ml, P less than 0.025; T: 0.55 +/- 0.17 ng/ml vs 0.62 +/- 0.19 ng/ml, P less than 0.05; E2: 32.95 +/- 19.63 pg/ml vs 66.98 +/- 54.83 pg/ml, P less than 0.001; LH/FSH: 0.91 +/- 0.44 vs 1.30 +/- 0.84, P less than 0.005) respectively. No significant difference was found between the two groups for F; however FSH levels correlated negatively with F levels only in the microfilaremia group (r = -0.38, n = 38, P less than 0.05). Our results suggest that the presence of microfilaremia in our subjects may have contributed to reduced steroid levels, perhaps by involvement of the cyclic AMP kinase system. These observations may explain the delayed menarche and androgen secretion found during puberty in a similar population living in the same zone of endemic filariasis. Microfilaremia should therefore be considered an environmental factor which mediates endocrine disorders in subjects living in tropical filariasis areas.