Two recent but temporally distinct outbreaks of cutaneous leishmaniasis among foreign workers in the Dead-Sea area of Jordan.

Two temporally distinct outbreaks of human cutaneous leishmaniasis (CL), as well as scattered cases of the disease, have recently been observed close to the Dead Sea, in Jordan. Each of the two outbreaks, which occurred in 2004/2005 and 2007/2008, involved a group of foreign workers who were deployed within otherwise uninhabited locations. During each outbreak, about 20% of the workers were found infected with the causative parasite. In the earlier outbreak, 61 workers were found to have skin lesions like those of CL and all but three were confirmed by culture and/or the examination of smears (40 cases) or, in the case of 18 (86%) of the 21 suspected cases found smear- and culture-negative, by PCR. In the second outbreak, the cases were only identified from their clinical manifestations and their response to antileishmanial treatment (cryotherapy). Leishmania major was identified as the cause of the 2004/2005 outbreak and some sporadic cases that occurred, in 2004, along the shores of the Dead Sea. The burrows of potential reservoir hosts were found close to the outbreak locations, frequently under the chenopod Seidlitzia rosmarinus. The two outbreaks emphasise the continuing problem posed by the CL focus in the Mid Jordan Valley and its impact on humans who move into the area. Curiously, an investigation on the socio-economic conditions of the workers during the outbreaks identified a group of 48 workers who were living in air-conditioned rooms during the 2007/2008 outbreak, among whom no CL cases were found. In contrast, 26 of a neighbouring group of 124 workers, who were all living in non-air-conditioned rooms, developed CL lesions. The role of air conditioning, and of other factors and measures, in the prevention of the transmission of the causative parasites of CL merits further investigation and the attention of the local health authorities.

Evaluation of PCR-RFLP (based on ITS-1 and HaeIII) for the detection of Leishmania species, using Greek canine isolates and Jordanian clinical material.

It is possible to detect and distinguish Leishmania parasites using PCR-RLFP - a combination of PCR and analysis of the fragment-length polymorphism seen when the amplicons are digested with one or more restriction enzymes. In the present study, clinical samples from 24 Jordanians suspected to have cutaneous leishmaniasis and cultures set up using leishmanial parasites from five Greek dogs were investigated using PCR, in which the internal-transcribed-spacer-1 (ITS1) region of the parasites' ribosomal-RNA gene was amplified, followed by HaeIII digestion of the resulting amplicons. The cultures, which were all maintained in Leibowitz L-15 medium with 20% foetal calf serum, were each investigated as serial dilutions. Using the PCR-RLFP analysis, each culture was identified as L. donovani and each was found positive for this parasite with a mean sensitivity of 66%-100% (depending on the culture dilution tested), a specificity of 100%, a mean positive predictive value of 100%, and a negative predictive value of 74.6%-100%. When simulated clinical samples, created by mixing human blood with known numbers of L. donovani promastigotes, were investigated, the PCR-RFLP gave optimal results (with a value of 100% each for sensitivity, specificity and positive and negative predictive values). When the real clinical samples (25 lesion aspirates and 20 samples of peripheral blood from 24 Jordanian patients) were investigated using the molecular method, 20 (84%) of the patients were found to have lesion aspirates that were PCR-RFLP-positive for L. major (although, by microscopy, only six were found to have amastigote-positive lesion aspirates). None of the blood samples from the Jordanian patients, however, was found PCR-positive.

Neisseria gonorrhoeae porin modulates phagosome maturation.

The porin (PorB) of Neisseria gonorrhoeae has been implicated in the pathogenesis of this species. Porin is believed to translocate from the bacterial outer membrane into target cell membranes affecting various cell functions. Here we investigated the effect of porin on phagosome maturation. Phagocytosis of latex beads by human macrophages was allowed in the presence or absence of purified porin. Isolation of latex bead-containing phagosomes and subsequent two-dimensional gel electrophoresis revealed substantial differences in the phagosomal protein composition. Immunoblotting detected higher amounts of annexin II and the early endocytic markers Rab5 and transferrin receptor and decreased levels of the late endocytic markers Rab7 and cathepsin D in phagosomes obtained in the presence of porin compared with those obtained in its absence. Furthermore, association of Rab4 with the latex bead-containing phagosomes was revealed by flow cytometry. The amount of this small GTPase was markedly higher in the phagosomes isolated in the presence of porin. The data thus indicate that neisserial porin is itself able to arrest phagosome maturation within macrophages.

Experimental infection of native human ureteral tissue with Neisseria gonorrhoeae: adhesion, invasion, intracellular fate, exocytosis, and passage through a stratified epithelium.

The exact mechanisms by which Neisseria gonorrhoeae invades the mucosal lining to cause local and disseminated infections are still not fully understood. The ability of gonococci to infect the human ureter and the mechanism of gonococcal infection in a stratified epithelium were investigated by using distal ureters excised from healthy adult kidney donors. In morphological terms, this tissue closely resembles parts of the urethral proximal epithelium, a site of natural gonococcal infection. Using piliated and nonpiliated variants of N. gonorrhoeae MS11, we demonstrated the importance of pili in the attachment of gonococci to native epithelial cells as well as their association with epithelial damage. By electron microscopy we elucidated the different mechanisms of colonization and invasion of a stratified epithelium, including adherence to surface cells, invasion and eventual release from infected cells, disintegration of intercellular connections followed by paracellular tissue infiltration, invasion of deeper cells, and initiation of cellular destruction and exfoliation resulting in thinning of the mucosa.

Serodiagnosis of cutaneous leishmaniasis in Jordan using indirect fluorescent antibody test and the enzyme-linked immunosorbent assay.

The usefulness of IFAT and ELISA, in the detection of antibodies to cutaneous leishmaniasis (CL) in Jordanian cases was studied. Serum samples were collected from three groups of confirmed or putative CL patients (n = 100), 132 healthy blood donors, 10 patients with pulmonary tuberculosis (TB), and 16 patients with typhoid fever (TF). Antigens for both tests were prepared from promastigotes of a Leishmania major isolate. At a serum dilution of respectively 1:16 and 1:100 both IFAT and ELISA had a sensitivity of 81%, whereas in the healthy control group their specificities were 95 and 96%. Maximal titers in the 37 parasitologically-proven cases were 1:128 in IFAT and 1:800 in ELISA. Antibodies were detected in about 50% of the 42 cases that had negative parasitological tests but had typical lesions with IFAT-titers up to 1:64 and ELISA titers up to 1:400. However, antibodies were detected in 19% of the 21 clinically-suspected cases of CL with maximal titers of 1:32 in IFAT and 1:200 in ELISA. A variation in antibody level was detected in the treated and the non-treated patients who were followed up for few months after diagnosis. One serum specimen taken from a patient with TB and two sera taken from patients with TF cross-reacted with Leishmania antigens in both IFAT and ELISA. This false positivity could be eliminated by absorption of these sera with their homologous antigens. There was no significant relationship between antibody level and duration of infection with CL. On the other hand, a significant relationship between antibody level and number of CL lesions was found. Although both tests would be useful for detection of circulating antibodies in cases suspected of having CL, especially in those having several lesions, IFAT is recommended for use in Jordan for its simplicity and rapidity.

Highly polarized primary urothelial cells from human ureter grown as spheroid-like vesicles.

Epithelial cells growing in vitro are frequently non-polarized and lack histophysiological characteristics. Furthermore, the quality of two-dimensional cell layers is limited by the physico-chemical properties of the support. Therefore, for an in vitro system to reflect the normal epithelial physiology, it is necessary to maintain the inner and outer geometrical configuration of the cells. In order to avoid the disadvantages of two-dimensional cultures we have established an in vitro model that closely resembles the in vivo situation. Human ureteral epithelial cells (HUEC) were used to prepare multicellular vesicles which maintain a geometrically intact cell organization that is not achieved in conventional cultures. Light and electron microscopy investigations showed the morphology of the cells to be similar to that in situ. HUEC vesicles are more in vivo-like than two-dimensional cultures and therefore represent a suitable model for a variety of research purposes including studies on the pathogenesis of micro-organisms.