Chikungunya virus and West Nile virus infections imported into Belgium, 2007-2012.

Arboviral infections are emerging among tourists travelling to (sub)tropical regions. This study aims to describe the importation of chikungunya virus (CHIKV) and West Nile virus (WNV) into Belgium over a 6-year period from 2007 to 2012. Clinical samples were obtained from travellers presenting at the outpatient clinic of the Institute of Tropical Medicine (ITM), Antwerp, Belgium or submitted to the Central Laboratory for Clinical Biology of the ITM. Testing was performed by serology and/or by real-time reverse transcriptase-polymerase chain reaction. A total of 1288 returning travellers were investigated for CHIKV infection resulting in 34 confirmed and two probable diagnoses (2·80%). Out of 899 patients, four confirmed and one probable imported WNV infections were diagnosed (0·55%). No locally acquired cases have been registered in Belgium until now and the geographical origin of the imported infections reflects the global locations where the viruses are circulating.

Heat-shock protein 70 gene sequencing for Leishmania species typing in European tropical infectious disease clinics.

We describe Leishmania species determination on clinical samples on the basis of partial sequencing of the heat-shock protein 70 gene (hsp70), without the need for parasite isolation. The method is especially suited for use in non-endemic infectious disease clinics dealing with relatively few cases on an annual basis, for which no fast high throughput diagnostic tests are needed. We show that the results obtained from this gene are in nearly perfect agreement with those from multilocus enzyme electrophoresis, which is still considered by many clinicians and the World Health Organization (WHO) as the gold standard in Leishmania species typing. Currently, 203 sequences are available that cover the entire hsp70 gene region analysed here, originating from a total of 41 leishmaniasis endemic countries, and representing 15 species and sub-species causing human disease. We also provide a detailed laboratory protocol that includes a step-by-step procedure of the typing methodology, to facilitate implementation in diagnostic laboratories.

Chikungunya infection confirmed in a Belgian traveller returning from Phuket (Thailand).

Chikungunya infection has been increasingly reported in international travellers following its epidemic re-emergence in the Indian Ocean islands in 2006 and its spread to southern Asia thereafter. We describe the first case of chikungunya in a Belgian traveller returning from Phuket, Thailand and discuss the potential implications of chikungunya cases imported to European countries for patient management and public health.

[Screening the asymptomatic traveler returning from the tropics]. / Bilan de santé chez le voyageur asymptomatique de retour des tropiques.

Travelers to developing countries may be exposed to several infectious pathogens sometimes leading to complications after a long latency period. Screening of asymptomatic returning travelers is only justified in case of specific risks during travel or after a long tropical stay (> 3 months). Based mainly on the history taking of risk patterns, some occult infections have to be looked for: tuberculosis, sexual transmitted infections and some parasitic conditions (schistosomiasis, amebiasis, strongyloidosis,...). Post-travel screening is most effective about 3 months after return, and may be even more beneficial if associated with screening of major western morbidities and preventive counseling for future travels.

Evaluation of two staging systems for HIV infection for use in developing countries.

OBJECTIVE: To evaluate the clinical axis of the World Health Organization (WHO) clinical staging system and the modified WHO staging system proposed by Montaner et al. using the lymphocyte strata > 1500, 1500-1000 and < 1000 cells x 10(6)/l. DESIGN: Cross-sectional study. PATIENTS: Four hundred and fifteen consecutive patients with HIV infection attending three HIV reference centres in Belgium. METHODS: Absolute CD4 lymphocyte counts were compared between stages within the two staging systems. RESULTS: Median CD4 lymphocyte counts decreased with increasing stage of disease in both staging systems. Differences in median CD4 lymphocyte counts between stages of each staging system were statistically significant (Kruskal-Wallis one-way analysis of variance, P < 0.001). The WHO clinical stage 1 and the modified WHO stage I had positive predictive values of 56 and 58%, respectively, for identifying patients with CD4 lymphocyte levels > 500 cells x 10(6)/l. The WHO clinical stage 4 and the modified WHO stage IV had positive predictive values of 79 and 80%, respectively, for identifying patients with CD4 lymphocyte levels < 200 cells x 10(6)/l. CONCLUSIONS: The WHO clinical staging system or a modified version of this system using lymphocytes stratification may be a good alternative in developing countries to the CD4 lymphocyte count-based HIV staging system used in the developed world. Cohort studies in developing countries are needed to assess their prognostic value.

PIP: In 1990, Belgium, physicians enrolled 415 consecutive patients attending HIV reference centers in Antwerp, Brussels, and Ghent in a cross-sectional study designed to evaluate the clinical axis of the WHO staging system with and without the lymphocyte stratification proposed by Montaner el al. (that is, modified WHO staging system) (1500, 1500- 1000, and 1000 cells x 1 million/l). They filled in a standardized questionnaire with all criteria of the WHO staging system. Laboratory personnel used standard hematology and flow cytometry techniques to determine absolute and CD4 lymphocyte counts. 80% of the patients were Caucasians. 46% of all patients were homosexual and 42% were heterosexual; 79.2% were men. Median CD4 lymphocyte counts fell in both staging systems as the stage of HIV infection increased. There were significant differences in median CD4 counts between stages of each staging system (p .001). The modified WHO staging system's stage I was more sensitive at identifying patients with CD4 lymphocyte counts of more than 500 cells x 1 million/l than the WHO clinical stage 1 (83% sensitivity vs. 48% sensitivity). The positive predictive value of WHO clinical stage 4 and of the modified WHO staging system's stage IV for identifying people with CD4 lymphocyte counts of less than 200 cells x 1 million/l was quite high (79% and 80%, respectively). The researchers suggested that clinicians use stages 4 and IV as end-points is clinical trials in developing countries. Clinicians completing the questionnaire knew the patients' earlier CD4 lymphocyte count, which may have introduced a bias in the study. For example, they may have more thoroughly examined patients with low CD4 lymphocyte counts than those with normal counts. Nevertheless, the study's results indicated that either one of these systems may be a good alternative in developing countries to the technical equipment-dependent CD4 lymphocyte count-based HIV staging system used in developed countries. Cohort studies in developing countries would evaluate their prognostic value.

A Belgian traveler who acquired yellow fever in the Gambia.

A 47-year-old Belgian woman acquired yellow fever during a 1-week vacation in The Gambia; she had never been vaccinated against yellow fever. She died of massive gastrointestinal bleeding 7 days after the onset of the first symptoms. This dramatic case demonstrates that it is important for persons to be vaccinated against yellow fever before they travel to countries where yellow fever is endemic, even if the country, like The Gambia, does not require travelers to be vaccinated.

Evaluation of two tests based on the detection of histidine rich protein 2 for the diagnosis of imported Plasmodium falciparum malaria.

The ParaSight-F dipstick test (Becton Dickinson, USA) and the ICT Malaria Pf test (ICT, Australia) both detect histidine rich protein 2 (HRP-2), a water-soluble antigen expressed by Plasmodium falciparum trophozoites. The present study compared the diagnostic performance of both tests in persons returning to Belgium from countries endemic for malaria. During a period of 18 months both tests were performed on all patients returning from the tropics with a positive malaria blood film. Patients with fever without an obvious cause were used as controls. For the ParaSight-F test, considering P. falciparum trophozoites only, sensitivity was 95% and specificity 90%. Considering trophozoites of all species of Plasmodium, sensitivity was 71% and specificity 87%. Finally, considering patients with clinical malaria, the sensitivity of the test was 72% and specificity 87%. For the ICT Malaria Pf test, sensitivity was 95% and specificity 89% for P. falciparum trophozoites only, 71% and 86% for trophozoites of all species, and 72% and 87% for clinical malaria. Both tests gave highly comparable results. However, antigen detection assays cannot replace conventional microscopy in diagnosing imported malaria. Thick blood film examination is more sensitive and more specific, it allows estimation of parasitaemia and distinction between parasite growth stages, and it covers all species. Moreover, with treated patients the use of antigen tests might lead to problems in determining the efficacy of therapy.

Treatment failure of a single high dose of ivermectin for Mansonella perstans filariasis.

Infections with Mansonella perstans are common in certain parts of Africa and South America. There is no standard treatment at present. We evaluated the effect of a single high dose of ivermectin (600 micrograms/kg) on microfilaraemia in 7 consecutive patients. No decrease in microfilarial counts could be demonstrated after a follow-up period of 7-56 d.

Primary targets for photoinactivation of vesicular stomatitis virus by AIPcS4 or Pc4 and red light.

Phthalocyanines are useful sensitizers for the photodynamic sterilization of red blood cell concentrates. The mechanism of photoinactivation of lipid-enveloped viruses is not completely understood. Vesicular stomatitis virus (VSV) was used as a model virus to study the primary targets of photoinactivation by aluminum phthalocyanine tetrasulfonate (AIPcS4) or silicon phthalocyanine HOSiPcOSi(CH3)2(CH2)3N(CH3)2 (Pc4) and red light. Inactivation conditions for VSV in buffer were determined using an end point dilution assay, and viral RNA synthesis in host cells was measured to determine the loss of infectivity in a direct way. The very rapid decrease in the viral RNA synthesis after photodynamic treatment was correlated with respect to different potential primary targets that are involved in different steps of the viral replication cycle. Damage to the viral proteins, induced by treatment with AIPcS4 or Pc4 and analyzed by gel electrophoresis, could not account for the observed loss of infectivity. Binding of VSV to host cells was only slightly impaired after photodynamic treatment with both sensitizers and could therefore not be responsible for the rapid decrease in viral RNA synthesis in cells. A very strong inhibition of viral RNA polymerase activity after treatment with AIPcS4 and red light was detectable using an in vitro assay. This decrease correlated well with the loss of infectivity, indicating that either the RNA or the viral RNA polymerase is the primary target for photoinactivation of VSV with AIPcS4. Treatment with Pc4 did not cause inhibition of viral RNA polymerase activity to an extent that could account for the observed very rapid loss of infectivity. It was therefore concluded that neither the viral proteins nor the binding to the host cells nor the RNA or RNA polymerase are the primary targets for photoinactivation of VSV by Pc4.

The effect of photodynamic treatment of yeast with the sensitizer chloroaluminum phthalocyanine on various cellular parameters.

Photodynamic treatment of Kluyveromyces marxianus with chloroaluminum-phthalocyanine resulted in loss of clonogenicity. Several parameters were studied to identify targets that could be related to loss of colony-forming capacity. Inhibition of various plasma membrane-bound processes was observed, such as substrate transport and plasma membrane ATPase activity. Moreover, K+ loss from the cells was observed. Photodynamic treatment also reduced the activity of various enzymes involved in energy metabolism, thereby decreasing the cellular ATP level. It will be discussed however that none of these processes is likely to be related directly to loss of clonogenicity. Treatment with phthalocyanine and light resulted in a strong inhibition of the incorporation of 14C-phenylalanine in trichloracetic acid-precipitable material. The induction of the beta-galactoside utilization system was also strongly inhibited. The latter two processes did not recover during incubation, subsequent to photodynamic treatment. It is concluded that photodynamically induced inhibition of protein synthesis is a critical factor contributing to the loss of clonogenicity.

Inhibition of various steps in the replication cycle of vesicular stomatitis virus contributes to its photoinactivation by AlPcS4 or Pc4 and red light.

Vesicular stomatitis virus (VSV) was used as a model virus to study the processes involved in photoinactivation by aluminum phthalocyanine tetrasulfonate (AlPcS4) or silicon phthalocyanine HOSiPcOSi(CH3)2(CH2)3N(CH3)2 (Pc4) and red light. Previously a very rapid decrease in the intracellular viral RNA synthesis after photodynamic treatment was observed. This decrease was correlated to different steps in the replication cycle. Binding of VSV to host cells and internalization were only slightly impaired and could be visualized by electron microscopy. The capability of the virus to fuse with membranes in an acidic endosomal environment was studied using both pyrene-labeled liposomes and a hemolysis assay as a model. These tests indicate a rapid decrease of fusion capacity after AlPcS4 treatment, which correlated with the decrease in RNA synthesis. For Pc4 treatment no such correlation was found. The fusion process is the first step in the replication cycle, affected by AlPcS4 treatment, but also in vitro RNA polymerase activity was previously shown to be inhibited. Inactivation of VSV by Pc4 treatment is apparently caused by damage to a variety of viral components. Photodynamic treatment of virus suspensions with both sensitizers causes formation of 8-oxo-7,8-dihydroguanosine in viral RNA as measured by HPLC with electrochemical detection. This damage might be partly responsible for inhibition of the in vitro viral RNA polymerase activity by photodynamic treatment.

Changing epidemiological and clinical aspects of imported malaria in Belgium.

BACKGROUND: In the early nineties the increase of imported malaria in some European countries was temporarily halted, but it resumed in 1994. More Africans, more European travelers, and fewer long-term residents were counted amongst patients. A shift towards more subacute disease has been noted. This study intends to assess whether the same trends were observed in Belgium. METHODS: Clinical and epidemiological data of 128 patients treated for malaria in 1997 at the Institute of Tropical Medicine and the University Hospital of Antwerp were compared with 209 malaria patients treated in 1988/1989. Risk factors for clinical presentation and parasitemia were analysed. RESULTS: In Belgium the number of reported imported malaria cases remained almost stable between 1988 and 1997. In 1997, there were more African patients, less infections from Central Africa, and 50% less residents. Less patients reported prophylaxis use. The causative agent shifted from Plasmodium falciparum to other species. Subacute and atypical malaria became less frequent. In both years, there were no deaths, and severe malaria did not increase significantly. Mefloquine disappeared almost as a curative treatment, and was replaced by quinine, with or without a long acting agent, or by halofantrine. The ethnic origin, nor the use of chemoprophylaxis, influenced disease characteristics. In 1988, malaria attacks in the previous months predisposed to subacute disease; longer residence, and attacks in the previous months, protected against high parasitemia; longer symptom duration correlated with absence of fever, and with splenomegaly. None of these risk factors was correlated with severe malaria. CONCLUSION: The incidence of subacute malaria dropped significantly in the last decade. Although this presentation is almost limited to residents, the decline in malaria can not be explained by an overall shorter duration of stay, since the decline in this particular clinical presentation of malaria was also spectacular in residents. Apparently, insufficient treatment of malaria attacks in the previous months is the only independent risk factor.

Single strand breaks and mutagenesis in yeast induced by photodynamic treatment with chloroaluminum phthalocyanine.

Photodynamic treatment of the yeast Kluyveromyces marxianus with the sensitizer aluminum phthalocyanine results in loss of clonogenicity. In this paper the effect of this treatment on DNA of this yeast was investigated by searching for single strand breaks and forward mutations. Using the alkaline step elution technique it was found that illumination of the yeast in the presence of aluminum phthalocyanine resulted in an increase in single strand breaks. These could, partially, be repaired by post-incubating illuminated cells in growth medium. At comparable survival levels, photodynamic treatment with aluminum phthalocyanine induced fewer single strand breaks than X-ray treatment. By using a medium containing 5-fluoroorotic acid, mutants in the uracil biosynthetic pathway were selected. Photodynamic treatment resulted in a light dose dependent increase of the mutation frequency. The observed mutagenicity of photodynamic treatment of the yeast with phthalocyanine was lower than the mutagenicity of UVC and X-ray treatment at equal colony forming capacity, indicating that photodynamic treatment is the least mutagenic of those treatments. It is concluded that photodynamic treatment of K. marxianus results in DNA damage. Saccharomyces cerevisiae rad14 and rad52 mutants were used to determine the effect of the nucleotide excision repair and recombinational repair pathways, respectively, on survival after photodynamic treatment. Our data indicate that DNA damage is not the main determinant for cell killing by photodynamic treatment and that the type of damage induced is apparently not subject to RAD14- or RAD52 controlled repair.

[Information communication to travelers, the French and Belgian experiences]. / Communication de l'information aux voyageurs, les expériences française et belge.

Providing relevant and up-to-date information to professionals and the general public is possible through many existing as well as developing means of communication including telephone and faxing, databases, answering machines and internet. Professionals may use information networks in order to harmonize advice given to their patients or clients. An exchange may also be established among all types of travel medicine professionals for keeping up with the latest relevant news and information or for sharing new ideas. Because travellers are now more proactive on their health than ever before, basic information designed for the general public is also becoming necessary. Examples of the various uses of communications in France and in Belgium are considered below.

[The principal arthropod vectors of disease. What are the risks of travellers' to be bitten? To be infected?]. / Les principaux arthropodes vecteurs de maladies. Quels risques pour le voyageur d'être piqué? D'être contaminé?

Many blood-sucking arthropods are potential vectors of disease. To become a vector, the arthropod must be susceptible to the infective agent and must survive the incubation period so as to transmit the pathogens to a host. While some arthropod associated diseases affect only man (e.g. malaria) most of these diseases are (anthropo-) zoonoses with man often an accidental host. The risk of contamination depends on the one hand on the biting behaviour of the vector, its biology and distribution, and on the other hand on the sites visited by the traveller, the length of his stay, his activities, the conditions of sleeping accommodation. The risk of contracting malaria is very high in tropical Africa, in the forest area of South America and South East Asia, in Papua New Guinea. Malaria can be prevented if measures (e.g. pyrethroid impregnated bed nets, repellents) are taken to avoid bites of Anopheline mosquitoes between sunset and sunrise, but appropriate chemoprophylaxis must not be neglected. Lethal cases of yellow fever among unvaccinated travellers still occur despite a strict international regulation on vaccination requirements. Dengue is a major health problem in intertropical areas. As no vaccine is available, personal protection measures are recommended against daytime-biting mosquitoes, including the use of protective clothing, repellents. Other arthropod borne diseases among travellers are less common but the risks increase during adventure trips (e.g. zoonotic leishmaniasis, tick-borne relapsing fever) and humanitarian actions (e.g. risk of louse-borne typhus during visits of overcrowded prisons). Tick-borne diseases receive nowadays more attention. These diseases are not only restricted to some occupations (farmers, veterinarians) but also ramblers and campers are at risk. Attached ticks should be removed rapidly and carefully, since several hours of attachment are needed for transmission of spirochetes of LYME disease.

Epidemiology and outcome of Shigella, Salmonella and Campylobacter infections in travellers returning from the tropics with fever and diarrhoea.

INTRODUCTION: During a study on fever after a stay in the tropics, we aimed at investigating the epidemiology and outcome of invasive bacterial enteritis due to Shigella, Salmonella or Campylobacter spp. in patients diagnosed with febrile traveller's diarrhoea. METHODS: From April 2000 to September 2006, we evaluated prospectively 594 travellers presenting with fever and diarrhoea within a month after a stay in the tropics. Patients not found with a systemic infection were assumed to have febrile traveller's diarrhoea (TD). Invasive bacterial enteritis was confirmed by isolation of Shigella, Campylobacter or nontyphoidal Salmonella in stool cultures. RESULTS: Systemic infections (mainly malaria) were diagnosed in 259 (44%) evaluated travellers. Invasive bacterial enteritis, either alone or with another infection, was confirmed in 114 (34%) of the 335 remaining patients with febrile TD. Aetiologies were distributed between Campylobacter jejuni (47, 41%), Shigella spp. (43, 38%), Salmonella spp. (22, 19%) and mixed Campylobacter-Salmonella infection (2, 2%). Invasive bacterial enteritis accounted for about a third of febrile TD cases occurring after a stay in sub-Saharan Africa, North Africa/Middle East or Latin America, and for half of those occurring after a travel to southern Asia (including 33% only due to C. jejuni). Resistance to fluoroquinolones was exclusively observed in C. jejuni isolates, but at an overall rate of 53%. Clinical failure occurred in 33% of the patients with C. jejuni infection empirically treated with a fluoroquinolone. CONCLUSION: Invasive bacterial enteritis was a frequent aetiology of febrile TD. C. jejuni was the leading pathogen after a travel to southern Asia, and was associated with high rate of resistance to fluoroquinolones and of clinical failure.