M. Van Esbroeck

The clinical spectrum of Exophiala jeanselmei, with a case report and in vitro antifungal susceptibility of the species.

Exophiala jeanselmei is clinically redefined as a rare agent of subcutaneous lesions of traumatic origin, eventually causing eumycetoma. Mycetoma is a localized, chronic, suppurative subcutaneous infection of tissue and contiguous bone after a traumatic inoculation of the causative organism. In advanced stages of the infection, one finds tumefaction, abscess formation and draining sinuses. The species has been described as being common in the environment, but molecular methods have only confirmed its occurrence in clinical samples. Current diagnostics of E. jeanselmei is based on sequence data of the Internal Transcribed Spacer (ITS) region of ribosomal DNA (rDNA), which sufficiently reflects the taxonomy of this group. The first purpose of this study was the re-identification of all clinical (n=11) and environmental strains (n=6) maintained under the name E. jeanselmei, and to establish clinical preference of the species in its restricted sense. Given the high incidence of eumycetoma in endemic areas, the second goal of this investigation was the evaluation of in vitro susceptibility of E.jeanselmei to eight conventional and new generations of antifungal drugs to improve antifungal therapy in patients. As an example, we describe a case of black grain mycetoma in a 43-year-old Thai male with several draining sinuses involving the left foot. The disease required extensive surgical excision coupled with intense antifungal chemotherapy to achieve cure. In vitro studies demonstrated that posaconazole and itraconazole had the highest antifungal activity against E. jeanselmei and E. oligosperma for which high MICs were found for caspofungin. However, their clinical effectiveness in the treatment of Exophiala infections remains to be determined.

Validation of a four-primer real-time PCR as a diagnostic tool for single and mixed Plasmodium infections

Although microscopy remains the reference standard for malaria diagnosis, molecular tools are attracting increasing interest. To improve the detection of mixed infections, we developed a four-primer real-time PCR with four Plasmodium species-specific forward primers, based on the pan-primer design with universal Plasmodium primers as described previously. After validation for analytical sensitivity, specificity and reproducibility, the four-primer PCR was evaluated on 351 blood samples from patients presenting at the outpatient clinic of the Institute of Tropical Medicine (Belgium). With the four-primer PCR, we identified 188 Plasmodium falciparum (Pf), 54 Plasmodium vivax (Pv), 52 Plasmodium ovale (Po) and 13 Plasmodium malariae (Pm) single infections, 27 mixed infections (14 Pf + Pm; 12 Pf + Po; one Pv + Pm) and 17 negative specimens. We found lower cycle threshold values than with the pan-primer PCR, with a mean difference of 2.23, a higher analytical sensitivity (in asexual parasites/μL: Pf/Pv, 0.02; Po, 0.004; Pm, 0.006) and 15 extra mixed infections. As compared with microscopy, 17 extra mixed infections were detected and Plasmodium species were identified in four microscopy-positive samples in which species identification was not possible. Additionally, the PCR corrected 13 species mismatches between Po and Pv, and in 11 cases detected Pf as a second species that was not identified by microscopy and in five of them was not detected by rapid diagnostic tests (RDTs). PCR confirmed the presence of Pf in 30/46 histidine-rich protein-2-positive samples that were microscopy-negative. We conclude that the presently developed four-primer real-time PCR is complementary to standard malaria diagnostic tests in clinical laboratories, with an added value for simultaneous identification of the four Plasmodium species and the detection of mixed infections.

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.

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.

Serological and genetic evidence for the presence of Seoul hantavirus in Rattus norvegicus in Flanders, Belgium

Seoul hantavirus (SEOV), carried by Rattus rattus (black rat) and R. norvegicus (Norway, brown rat), was reported to circulate as well as cause HFRS cases in Asia. As Rattus sp. are present worldwide, SEOV has the potential to cause human disease worldwide. In Europe however, only SEOV prevalence in rats from France was reported and no confirmed cases of SEOV infection were published. We here report genetic and serological evidence for the presence of SEOV virus in brown rat populations in Belgium. We also serologically screened an at-risk group that was in contact with R. norvegicus on a daily basis and found no evidence for SEOV infection.

Syphilis reinfections pose problems for syphilis diagnosis in Antwerp, Belgium - 1992 to 2012

Persons with multiple syphilis reinfections may play an important role in syphilis transmission. We analysed all syphilis tests carried out for people attending the HIV/sexually transmitted infection (STI) clinic at the Institute of Tropical Medicine, Antwerp, Belgium, from 1992 to 2012 to evaluate the extent to which syphilis reinfections were contributing to the syphilis epidemic in Antwerp. We then characterised the features of the syphilis infections in individuals with five or more episodes of syphilis. A total of 729 syphilis episodes were diagnosed in 454 persons. The majority of syphilis episodes occurred in people who had more than one episode of syphilis (445/729; 61%). A total of 10 individuals had five or more episodes of syphilis diagnosed over this period. All were men who have sex with men, HIV positive and on antiretroviral therapy. They had a total of 52 episodes of syphilis diagnosed and treated. In 38/42 of the episodes of repeat syphilis in these 10 individuals, they presented without any signs or symptoms of syphilis. Given that the majority of cases of incident syphilis in our clinic were persons with reinfections and that they frequently presented without signs of symptoms of syphilis, there is a strong case for frequent and repeated screening in all persons with a diagnosis of syphilis.

Selective ambulatory management of imported falciparum malaria: a 5-year prospective study

The ambulatory management of imported Plasmodium falciparum malaria is controversial because criteria for safe selection of patients are imprecise. The aim of the present study was to investigate the evolution and outcome of patients diagnosed with Plasmodium falciparum malaria at a Belgian referral institute in order to assess the safety of the institute’s current selective ambulatory management protocol. From 2000 to 2005, all patients diagnosed with P. falciparum infection at the Institute of Tropical Medicine and the University Hospital of Antwerp were enrolled prospectively. Ambulatory treatment was offered to nonvomiting patients if they exhibited none of the 2000 World Health Organization criteria of severity and had parasitemia below 1% at the initial assessment. The treatment of choice was quinine (plus doxycycline or clindamycin) for inpatients and atovaquone-proguanil for outpatients. P. falciparum malaria was diagnosed in 387 patients, of whom 246 (64%) were Western travelers or expatriates and 117 (30%) were already on antimalarial therapy. At diagnosis, 60 (15%) patients had severe malaria. Vital organ dysfunction was initially seen in 34 and developed later in five others. Five patients died. Of the 327 patients initially assessed as having uncomplicated malaria, 113 (35%) were admitted immediately; of these, 4 developed parasitemia ≥5% at a later stage but without any clinical consequence. None of the 214 individuals initially treated as outpatients experienced any malaria-related complications, including 10 who were admitted later. Vital organ dysfunction was observed in only 2 of the 214 patients with initial parasitemia <1% who had not taken antimalarial agents (both patients had impaired consciousness at presentation). Ambulatory treatment is safe in treatment-naive malaria patients with parasitemia <1% who do not vomit and who do not exhibit any criteria of severe malaria.

Concomitant leptospirosis-hantavirus co-infection in acute patients hospitalized in Sri Lanka: implications for a potentially worldwide underestimated problem.

Two global (re-)emerging zoonoses, leptospirosis and hantavirus infections, are clinically indistinguishable. Thirty-one patients, hospitalized in Sri Lanka for acute severe leptospirosis, were after exclusion of other potentially involved pathogens, prospectively screened with IgM ELISA for both pathogens. Of these, nine (29·0%) were positive for leptospirosis only, one (3·2%) for hantavirus only, seven (22·5%) for both pathogens concomitantly, whereas 13 (41·9%) remained negative for both. Moreover, in a retrospective study of 23 former patients, serologically confirmed for past leptospirosis, six (26·0%) were also positive in two different IgG ELISA hantavirus formats. Surprisingly, European Puumala hantavirus (PUUV) results were constantly higher, although statistically not significantly different, than Asian Hantaan virus (HTNV), suggesting an unexplained cross-reaction, since PUUV is considered absent throughout Asia. Moreover, RT-PCR on all hantavirus IgM ELISA positives was negative. Concomitant leptospirosis-hantavirus infections are probably heavily underestimated worldwide, compromising epidemiological data, therapeutical decisions, and clinical outcome.

EPIDEMIOLOGY AND OUTCOME OF SHIGELLA, SALMONELLA AND CAMPYLOBACTER INFECTIONS IN TRAVELLERS RETURNING FROM THE TROPICS WITH FEVER AND DIARRHOEA

Abstract 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.

Evaluation of the Immunoquick+4 malaria rapid diagnostic test in a non-endemic setting

The aim of this retrospective study was to evaluate the Immunoquick+4 (BioSynex, Strasbourg, France), a three-band malaria rapid diagnostic test (MRDT) targeting histidine-rich protein-2 (HRP-2) and pan Plasmodium-specific parasite lactate dehydrogenase, in a non-endemic reference setting. Stored whole-blood samples (n = 613) from international travellers suspected of malaria were used, with microscopy corrected by polymerase chain reaction (PCR) as the reference method. Samples infected by P. falciparum (n = 323), P. vivax (n = 97), P. ovale (n = 73) and P. malariae (n = 25) were selected, as well as 95 malaria-negative samples. The overall sensitivities of the Immunoquick+4 for the diagnosis of P. falciparum, P. vivax, P. malariae and P. ovale were 88.9, 75.3, 56.0 and 19.2%, respectively. Sensitivity was significantly related to parasite density for P. falciparum (93.6% versus 71.4% at parasite densities >100/µl and ≤100/µl, respectively) and P. vivax (86.8% versus 48.3% at parasite densities >500/µl and ≤500/µl, respectively). The Immunoquick+4 showed good reproducibility and reliability for both test results and line intensities. The Immunoquick+4 performed well for the detection of P. falciparum and P. vivax.