Monitoring the response of patients with cutaneous leishmaniasis to treatment with pentamidine isethionate by quantitative real-time PCR, and identification of Leishmania parasites not responding to therapy.

BACKGROUND: Leishmania (Viannia) guyanensis is believed to be the principal cause of cutaneous leishmaniasis (CL) in Suriname. This disease is treated with pentamidine isethionate (PI), but treatment failure has increasingly been reported. AIM: To evaluate PI for its clinical efficacy, to compare parasite load, and to assess the possibility of treatment failure due to other infecting Leishmania species. METHODS: Parasite load of patients with CL was determined in skin biopsies using real-time quantitative PCR before treatment and 6 and 12 weeks after treatment. Clinical responses were evaluated at week 12 and compared with parasite load. In parallel, molecular species differentiation was performed. RESULTS: L. (V.) guyanensis was the main infecting species in 129 of 143 patients (about 90%). PI treatment led to a significant decrease (P < 0.001) in parasite counts, and cured about 75% of these patients. Treatment failure was attributable to infections with Leishmania (Viannia) braziliensis, Leishmania (Leishmania) amazonensis and L. (V.) guyanensis (1/92, 1/92 and 22/92 evaluable cases, respectively). There was substantial agreement beyond chance between the parasite load at week 6 and the clinical outcome at week 12, as indicated by the κ value of 0.61. CONCLUSIONS: L. (V.) guyanensis is the main infecting species of CL in Suriname, followed by L. (V.) braziliensis and L. (L.) amazonensis. Furthermore, patient response to PI can be better anticipated based on the parasite load 6 weeks after the treatment rather than on parasite load before treatment.

Tolerability and efficacy of hydrocolloid dressings in the treatment of venous leg ulcers under tropical conditions: an open prospective study.

In the Western world, hydrocolloid dressings are widely used in wound treatment. However, little is known about their tolerability and efficacy under tropical conditions. The purpose of this study was to assess the tolerability and efficacy of a hydrocolloid dressing in combination with short stretch compressive bandages under tropical conditions. Seventeen patients with venous leg ulcers attending an outpatient clinic in Surinam were enrolled in the study for a period of 6 weeks. Swabs for bacterial cultures were taken at the beginning and end of the study. All ulcers showed a good healing tendency. Percentage of granulation tissue in the ulcers improved from mean 27% at start to 92% at the end. Mean circumference at start was 9.9 cm, at the end 4.9 cm. Exudation diminished from moderate in six and severe in eight ulcers, to moderate in 10 and almost none in two ulcers. In general, the dressing was very well accepted, pain was never reported. Leakage was noticed 39 times in the 164 dressing changes. This study revealed no differences in the rate of bacterial infections or colonization of wounds compared with studies performed in temperate regions. Our data indicate that hydrocolloid dressings can be used under tropical conditions.

Latex based, rapid and easy assay for human leptospirosis in a single test format.

Leptospirosis is an often severe disease which requires prompt treatment. Laboratory testing is required to reach a valid diagnosis. An agglutination assay for the detection of Leptospira-specific antibodies consisting of individually wrapped agglutination cards containing a stable, dried detection reagent is evaluated. The assay is simply performed by suspending the dried reagent with a drop of serum. The result is obtained within 30 s. The sensitivity of the assay varied with the stage of the disease and was 72.3% for samples collected during the first 10 days of the illness and 88.2% for samples collected at a later stage. The specificity was 93.9% and 89.8%, respectively. These characteristics make the test ideal for use in areas where the disease is common and where laboratory support is not routinely available.

International multicenter evaluation of the clinical utility of a dipstick assay for detection of Leptospira-specific immunoglobulin M antibodies in human serum specimens.

We performed a multicenter evaluation of a robust and easily performed dipstick assay for the serodiagnosis of human leptospirosis. The assay is aimed at the detection of Leptospira-specific immunoglobulin M (IgM) antibodies. The study involved 2,665 serum samples collected from 2,057 patients with suspected leptospirosis in 12 countries on five continents with different levels of endemicity and different surveillance systems. The patients were grouped as laboratory-confirmed leptospirosis case patients and noncase patients based on the results of culturing and the microscopic agglutination test. Paired samples from 27.7% of the subjects were tested. Of the 485 case patients, 87.4% had a positive dipstick result for one or more samples. Of the 1,513 noncase patients, only 7.2% had a positive result. Whereas most (88.4%) of the positive samples from the case patients showed moderate to strong (2+ to 4+) staining in the dipstick assay, most (68.1%) of the positive samples from the noncase patients showed weak (1+) staining. The sensitivity of the dipstick assay increased from 60.1% for acute-phase serum samples to 87.4% for convalescent-phase samples. The specificities for these two groups of samples were 94.1 and 92.7%, respectively. The dipstick assay detected a broad variety of serogroups. The results of the dipstick assay were concordant (observed agreement, 93.2%; kappa value, 0.76) with the results of an enzyme-linked immunosorbent assay for the detection of specific IgM antibodies, a test which is often used in the laboratory diagnosis of current or recent leptospirosis. This study demonstrated that this easily performed dipstick assay is a valuable and useful test for the quick screening for leptospirosis; has a wide applicability in different countries with different degrees of endemicity; can be used at all levels of the health care system, including the field; and will be useful for detecting and monitoring outbreaks of leptospirosis.

Mycobacterium leprae-specific T cells from a tuberculoid leprosy patient suppress HLA-DR3-restricted T cell responses to an immunodominant epitope on 65-kDa hsp of mycobacteria.

The polar tuberculoid type (TT) of leprosy, characterized by high T cell reactivity to Mycobacterium leprae, is associated with HLA-DR3. Surprisingly, DR3-restricted low T cell responsiveness to M. leprae was found in HLA-DR3-positive TT leprosy patients. This low responsiveness was specifically induced by M. leprae but not by M. tuberculosis and was seen only in patients and not in healthy controls. We studied this patient-specific, M. leprae-induced, DR3-restricted low T cell responsiveness in depth in one representative HLA-DR3-positive TT leprosy patient by using T cell clones. From this patient two types of T cell clones were obtained: one type was cross-reactive with M. tuberculosis and recognized an immunodominant epitope (amino acids 3 to 13) on the 65-kDa heat shock protein (hsp) the other type was M. leprae specific and reacted to a protein other than the 65-kDa one. To examine whether these M. leprae-specific T cell clones were responsible for the DR3-restricted low responsiveness to M. leprae, we tested them for the ability to suppress the proliferation of the DR3-restricted, 65-kDa, hsp-reactive clones. The DR3-restricted, M. leprae-specific T cells completely suppressed the proliferative responses of DR3-restricted, cross-reactive T cell clones to the 65-kDa hsp from the same patient as well as from other individuals. Also, DR3-restricted responses to an irrelevant Ag were suppressed by the M. leprae-specific T cell clones. However, no suppression of non-DR3-restricted T cell responses was seen. Although the mechanism must still be elucidated, this M. leprae-induced, DR3-restricted immunosuppression may at least partly explain the observed DR3-associated low T cell responsiveness in TT leprosy patients.

In vitro synthesis of antimycobacterial antibodies with different specificities in various tissues of leprosy patients.

For the detection of the synthesis in vitro of anti-Mycobacterium leprae antibodies in various tissues of leprosy patients, biopsy specimens of skin lesions, nasal mucosa, larynx, lymph nodes, and bone marrow were cultured in a medium containing 14C-labeled lysine and isoleucine. The culture fluids were analyzed by crossed immunoelectrophoresis with intermediate gel and autoradiography. The results show that synthesis of anti-M. leprae antibodies occurs at the investigated sites of leprosy patients and that the specificities of the synthesized antibodies differ between sites in individual patients. It is conceivable that these antibodies play a role in the local defense against M. leprae.

Immune complexes in leprosy patients from an endemic and a nonendemic area and a longitudinal study of the relationship between complement breakdown products and the clinical activity of erythema nodosum leprosum

The sera from leprosy patients living in Surinam (endemic) and 41 leprosy patients living in The Netherlands (nonendemic) were tested for circulating immune complexes (CIC) with the Clq-binding assay and the conglutinin-binding assay and for complement.

In vitro synthesis of anti-mycobacterial antibodies in biopsies from skin lesions of leprosy patients.

To demonstrate local synthesis of anti-Mycobacterium leprae antibodies, biopsies from skin lesions of leprosy patients were cultured in vitro in a medium containing 14C-labeled lysine and isoleucine, and the culture fluids were analyzed by crossed immunoelectrophoresis with intermediate gel and autoradiography. The results show that anti-M. leprae antibodies were synthesized in vitro in the biopsies from the skin lesions of leprosy patients and that the specificity of the locally produced antibodies varied from patient to patient.

In vitro synthesis of humoral factors (immunoglobulins and complement) in lesional skin of leprosy patients.

An in vitro culture technique was used to demonstrate the synthesis of immunoglobulins and complement in lesional skin of patients representing the entire clinico-histopathological spectrum of leprosy. The results indicate that immunoglobulin G is produced in different amounts in the various forms of leprosy. Classification of the patients according to the three main groups shows that a small amount of immunoglobulin G synthesis occurred in tuberculoid leprosy, a distinct amount occurred in borderline leprosy, and large amount occurred in lepromatous leprosy. Contrary to expectation, synthesis of C3 was found only in some of the cultures of these three forms of leprosy. The function of the locally synthesized immunoglobulin G and the findings concerning C3 synthesis are discussed.

In vitro synthesis of immunoglobulins, secretory component, complement and lysozyme by human gastrointestinal tissues. II. Pathological tissues.

An in vitro culture technique has been used to study synthesis of proteins by biopsies of human gastrointestinal mucosa which were obtained at endoscopy or surgery from patients with biliary gastritis, atrophic gastritis, peptic ulcer, gastric cancer, coeliac disease, Crohn's disease and ulcerative colitis. As in normal mucosa, immunoglobulin synthesis was found in all sites, but marked increases, especially in IgG, were seen in biliary gastritis and ulcerative colitis. In untreated coeliac disease, synthesis of IgG and IgM was increased. Synthesis of complement components did not differ from that found in normal mucosa. Increased lysozyme synthesis was seen in Crohn's disease. This study shows that useful information may be acquired from short-term culture studies of the small biopsies obtained with fibre optic endoscopes.

In vitro synthesis of immunoglobulins, secretory component, complement and lysozyme by human gastrointestinal tissues. I. Normal tissues.

An in vitro culture technique has been used to demonstrate synthesis of proteins by human gastrointestinal tissues cultured in vitro. Histologically normal tissues were obtained endoscopically and surgically. IgA and secretory component (SC) were produced in all sites, but the relative intensity of IgA synthesis and SC synthesis varied. In stomach and small intestine the intensity of IgA synthesis was greater than that of SC, but in large bowel mucosa, there appeared to be an excess of SC synthesis. Synthesis of IgG and IgM was also found in all sites. Complement proteins were produced by some of the intestinal biopsies, and by parotid gland. Lysozyme was synthesized by parotid gland and by gastric mucosa, and to a lesser extent in small intestine, and rarely in large intestine. The results suggest that in addition to the local mucosal IgA system the local production of other immunoglobulins, as well as non-immunoglobulin humoral defence factors, may be important host defences of the normal gastrointestinal tract.

In vitro synthesis of some complement components (C1q, C3 and C4) by lymphoid tissues and circulating leucocytes in man.

Human lymphoid tissues and peripheral blood leucocytes and monocytes were studies with respect to the synthesis of complement components (C1q, C3 and C4) using an in vitro culture technique. All of the lymphoid tissues investigated (bone marrow, thymus, lymph node, spleen, tonsil, adenoid) synthesize complement components in different patterns. C3 was produced by all lymphoid tissues except the spleen, which was the only lymphoid tissue in which C4 production was regularly found. C1q synthesis was demonstrated in the spleen and adenoid cultures, and occasionally also in those of lymph node tissue. Lymphocytes in peripheral blood from normal individuals and in thoracic duct lymph, and also from patients suffering from chronic lymphatic leukaemia, do not synthesize any of these complement components. Peripheral blood leucocyte samples from normal individuals, containing 60 per cent lymphocytes and 40 per cent monocytes, do synthesize C3, however. Separation of the monocytes from these samples showed that it was in these cells that the synthesis of C3 occurred. Production of C3 by mononuclear phagocytes is also supported by the finding that peripheral blood leucocytes from patients suffering from acute monocytic leukaemia synthesize C3. C1q and C4 synthesis could not be demonstrated in any of the cultures of circulating leucocytes.