Michael Kemp

Excretion of ciprofloxacin in sweat and multiresistant Staphylococcus epidermidis

BACKGROUND Staphylococcus epidermidis develops resistance to ciprofloxacin rapidly. That this antibiotic is excreted in apocrine and eccrine sweat of healthy individuals might be the reason for the development of such resistance. We assessed whether S epidermidis isolated from the axilla and nasal flora of healthy people could develop resistance to ciprofloxacin after a 1-week course of this antibiotic. METHODS The concentration of ciprofloxacin in sweat was measured in seven volunteers after oral administration of 750 mg ciprofloxacin twice daily for 7 days, and the development of resistance in S epidermidis from axilla and nostrils was monitored during and 2 months after the treatment. Genotyping of S epidermidis was done by restriction fragment length polymorphism. FINDINGS The mean concentration of ciprofloxacin in sweat increased during the 7 days of treatment-from 2.2 micrograms/mL 2.5 h after the first tablet to 2.5 micrograms/mL after the fifth tablet, and 5.5 micrograms/mL after the 13th tablet. All persons harboured susceptible S epidermidis (minimal inhibitory concentration [MIC] 0.25 microgram/mL) in axilla and nostrils before treatment. Four resistant strains were detected, two intermediate-level (MIC 4-12 micrograms/mL) and two high-level (MIC > 32 micrograms/mL). Three of these strains were found in all the participants, and a ciprofloxacin-sensitive variant of one of the high-level resistant strains was also found before the start of the treatment. The high-level resistant strains were also resistant to methicillin, erythromycin, gentamicin, sulphonamide, and trimethoprim. A mean of 2.7 days after the start of the treatment, development of ciprofloxacin resistance was detected in S epidermidis from the axilla of all persons, compared with 11 days for the appearance of resistant S epidermidis in nostrils. The resistant strains persisted for an average of 37 and 39 days in axilla and nostrils, respectively, after the end of the treatment. INTERPRETATION The rapid development of resistance to ciprofloxacin due to excretion of this drug into the sweat might be involved in the development of multiresistant S epidermidis and possibly other skin bacteria in hospitals and in communities with high use of ciprofloxacin or related drugs.

Dichotomy of the human T cell response to Leishmania antigens. I. Th1-like response to Leishmania major promastigote antigens in individuals recovered from cutaneous leishmaniasis

The T cell response to antigens from Leishmania major promastigotes was investigated in peripheral blood mononuclear cells from Sudanese individuals with a history of cutaneous leishmaniasis (CL), Sudanese individuals with positive DTH reaction in the leishmanin skin test but with no history of skin lesions, and in Danes without known exposure to Leishmania parasites. Proliferation and production of interferon‐gamma (IFN‐γ) and IL‐4 in antigen‐stimulated cultures was measured. Lymphocytes from individuals with a history of CL proliferated vigorously and produced IFN‐γ after stimulation with either a crude preparation of L. major antigens or the major surface protease gp63. These cultures produced no or only little IL‐4. Also cells from leishmanin skin test‐positive donors with no history of CL produced IFN‐γ and no IL‐4 in response to major antigens. Cells from the unexposed Danes were not activated by gp63. The cells from Danish donors produced either IFN‐γ or IL‐4, but not both cytokines after incubation with the crude preparation of L. major antigens. The data show that the T cell response to Leishmania antigens in humans who have had uncomplicated CL or subclinical L. major infection is an IFN‐γ‐producing Th1‐like response.

Dichotomy of the human T cell response to Leishmania antigens. II. Absent or Th2-like response to gp63 and Th1-like response to lipophosphoglycan-associated protein in cells from cured visceral leishmaniasis patients.

The T cell response to different Leishmania donovani aniigens was investigated using peripheral blood mononuciear cells (PBMC) from Kenyans cured of visceral leishmaniasis and non‐exposed Danes. Crude promastigote and amastigote antigens both induced proliferation and interferon‐gamma (IFN‐γ) production in PBMC from cured patients, while cells from non‐exposed donors gave weak responses. A similar pattern was indticed by lipophosphoglycan‐associated protein (LPGAP). By contrast, the major surface protease of Leishmania. gp63, induced only a weak proliferative response without IFN‐γ production in five of 17 samples from cured patients. Four of the five responding cultures produced IL‐4, i.e. the response to this antigen was of the Th2 type. Furthermore, sera from acutely ill visceral leishmaniasis patients contained high levels of IgG antibodies to gp63. The Th2‐tike response to gp63 in patients cured of visceral leishmaniasis differs from the Thl‐like response to the same antigen observed in patients cured of cutaneous leishmaniasis.

Dichotomy of the T cell response to Leishmania antigens in patients suffering from cutaneous leishmaniasis; absence or scarcity of Th1 activity is associated with severe infections

The T cell response was studied in 25 patients suffering from cutaneous leishmaniasis caused by Leishmania major with severe (n= 10) and mild (n= 15) disease manifestations. Peripheral blood mononuclear cells (PBMC) from the patients were activated by sonicates of Leishmania promastigotes (LMP) and amastigotes (LDA), and the surface protease gp63. The proliferative responses to Leishmania antigens were lower in patients with severe disease than in patients with mild disease (P= 0·01–0·05), and such a difference was not observed in the response to purified protein derivative of tuberculin (PPD) or tetanus toxoid (TT). LMP‐induced interferon‐gamma (IFN‐γ) production was lower in patients with severe than in patients with mild disease (P < 0·05). When the IL‐4 and IFN‐γ responses of each patient were considered, two response patterns were observed in the cultures activated by the Leishmania sonicates. One response pattern was characterized by high production of IFN‐γ without production of IL‐4 (a Th1‐like pattern), the other was characterized by low IFN‐γ levels which in most cases were associated with IL‐4 production (not a Th1‐like pattern). These patterns could not be distinguished when the cells from the same donors were stimulated by TT and PPD. The percentages of patients with a Th1‐like response pattern after stimulation by LMP in patients with severe and mild disease manifestations were 30% and 80%, respectively. This difference was statistically significant (P= 0·034).

Interleukin-4 and interferon-gamma production by Leishmania stimulated peripheral blood mononuclear cells from nonexposed individuals.

Interferon‐gamma (IFN‐γ) and interleukin‐4 (IL‐4) production by Leishmania reactive peripheral blood mononuclear cells (PBMC) from non‐exposed individuals was investigated. IFN‐γ was measured in culture supernatants after antigen stimulation. For the measurement of IL‐4, antigen stimulated cells were pulsed with PMA and ionomycin before IL‐4 release was measured. L. donovani and L. major antigens induced IL‐4 production (105–1748pg/ml) in 13 and seven cultures, and IFN‐γ production (1.7‐ > 66IU/ml) in 14 and 11 of 20 cultures, respectively. IL‐4 production rose steeply after 6 days of antigen stimulation suggesting a response due to antigen recognition. Both IL‐4 and IFN‐γ production was abrogated by depletion of CD2+ or CD4+ but not CD8+ cells. CD2+ or CD4+ but not CD8+ enriched cultures produced cytokines as unseparated PBMC. Thus, in non‐exposed individuals circulating Leishmania reactive CD4+ T cells could be demonstrated. The cells from different individuals showed different patterns of IFN‐γ and/or IL‐4 production upon antigenic stimulation. In experimental leishmaniasis the early balance between IFN‐γ and IL‐4 is important for the clinical outcome. Our findings call for studies of the importance of cytokine production by cross‐reactive T cells for the outcome of L. donovani infections in humans and show that the method for IL‐4 detection is useful for this purpose.

Diagnosis of cystic fibrosis related diabetes: a selective approach in performing the oral glucose tolerance test based on a combination of clinical and biochemical criteria

BACKGROUND Cystic fibrosis related diabetes (CFRD) has become increasingly common with the increasing longevity of patients with cystic fibrosis. The diagnosis of CFRD is important as its development may lead to a clinical deterioration which may be reversed with treatment. The oral glucose tolerance test (OGTT) is the method of choice in the diagnosis of CFRD, but performing OGTTs on all patients is inconvenient for patients and labour intensive for staff. The aim of this study was to identify a more selective approach in performing OGTTs in the diagnosis of CFRD based on the use of a combination of clinical and biochemical criteria. METHODS Clinically stable adult patients with cystic fibrosis not known to be diabetic attending the Royal Brompton Hospital Cystic Fibrosis Clinic for their annual review were invited to return within a month to have an OGTT. The result of the OGTT was compared with the results of tests performed during the annual review. The sensitivities and specificities of various methods used in the screening or diagnosis of CFRD were determined using OGTT as the “gold standard” diagnostic method. The combination of clinical and biochemical criteria which resulted in the highest sensitivity and specificity in the diagnosis of CFRD was determined. RESULTS Between August 1996 and May 1997 122 patients became eligible for the study, 91 of whom agreed to take part. The number of patients with normal, impaired, and diabetic glucose tolerance was 58 (64%), 21 (23%), and 12 (13%), respectively. When used alone, abnormal glycosylated haemoglobin (HbA1c) was found to have the highest sensitivity (83%; 95% CI 62 to 100) in the diagnosis of CFRD. The combination of an abnormal random blood glucose and/or abnormal HbA1c and/or symptoms of hyperglycaemia or weight loss was found to have the highest sensitivity (92%; 95% CI 76 to 100) in the diagnosis of CFRD. The specificity of this combination in the diagnosis of CFRD was 79% (95% CI 70 to 88). By selectively performing OGTTs in patients with one or more of the criteria cited above, 11 of the 12 patients with OGTT defined diabetes would have been identified. CONCLUSIONS Patients with cystic fibrosis already have to undergo a large number of routine investigations. The selective approach in performing OGTTs described here has the potential to identify the majority of patients with CFRD without the need to perform this investigation on all patients. This approach is likely to be welcomed by patients and will lead to significant savings in terms of time and resources for patients and staff. Further larger studies are warranted to validate this selective approach in the diagnosis of CFRD.

Regulator and effector functions of T‐cell subsets in human Leishmanaia infections

Because of an increasing number of patients suffering from Leishmania infections and because of the serious consequences of these infections more thorough knowledge of the host factors responsible for resistance and susceptibility to the diseases is needed. In murine models of Leishmania infections the cytokine production by CD4+ T cells has been identified as a major factor in determining the outcome of the infection. In these models Th1 cells producing IFN-gamma provide protection against the infection whereas Th2 cells producing IL-4 and IL-10 aggravate the disease. The fatal outcome of Leishmania infections in humans with defects in T-cell functions illustrates that these cells are fundamental in the defence against Leishmania in humans also. However, as for many other infectious diseases (meningococcal disease and other septicaemic conditions, pneumonia, viral hepatitis, schistosomiasis) the immune reactions to Leishmania parasites in humans can be associated with both protection and pathogenesis. Many individuals without previous exposure to Leishmania parasites have T cells which can respond to Leishmania antigens. These cells have the potential to generate either Th1 or Th2 like responses. During infection with Leishmania parasites humans develop specific T-cell recognition of well-characterized parasite antigens. T cells producing disease-exacerbating factors such as IL-4 in response to Leishmania antigen stimulation have been identified in humans as well as in mice. Both Th1 like and Th2 like cells recognizing Leishmania antigens can be expanded during infection. At the polyclonal level Th1 like responses to Leishmania antigens are found in individuals who have had self-healing or asymptomatic infections. Factors secreted by such Leishmania specific Th1 like cells can induce killing of intracellular parasites in infected macrophages. In individuals who have been cured from uncontrollable disseminating disease both Th1 and Th2 like responses can be detected. A restriction in the antigen recognition to particular protein fractions could not be demonstrated in the Th1 or Th2 like responses. These findings suggest an association between the pattern of cytokines produced by parasite specific T cells and the clinical course of the infection similar to the one seen in mice. In the murine model the cytokine pattern present in the animal at the time of infection can determine whether a Th1- or a Th2 response will develop. In vitro studies on human and murine cells have confirmed that certain cytokines (e.g. IFN-gamma, IL-12) will favour maturation of Th1 responses whereas others (e.g. IL-4, IL-10) support Th2 development. If similar immunoregulatory mechanisms operate in mouse and man, design of vaccines against human leishmaniasis should aim at introducing powerful Th1 like responses. Importantly, once generation of either Th1 or Th2 has started, the immune response seems to be locked in this pattern, even when it is harmful to the host. Therefore new vaccines against leishmaniasis should be designed in a way that they generate controlled Th1 like primary responses.

Production of interferon-gamma and interleukin-4 by human T cells recognizing Leishmania lipophosphoglycan-associated protein

The Leishmania protein LPGAP which is co-isolated with lipophosphoglycan is a specific activator of T cells from individuals who have recovered from American leishmaniasis. We have tested the effect of LPGAP on peripheral blood mononuclear cells (PBMC) from Kenyan donors cured from L. donovani infections. LPGAP induced vigorous proliferation and production of interferon-gamma (IFN-gamma) by the cells. In addition PBMC incubated with LPGAP released interleukin-4 (IL-4) after pulsing with ionomycin and phorbol myristate acetate. Single cells were isolated from LPGAP-stimulated cell lines and expanded as T-cell clones. LPGAP-reactive T-cell clones were activated by crude preparations of both promastigotes and axenic grown amastigote-like parasites. Among 9 CD4+ T-cell clones recognizing LPGAP, cells secreting predominantly IFN-gamma as well as cells secreting predominantly IL-4 were identified. The results show that both IFN-gamma producing (Th1-like) and IL-4 producing (Th2-like) T cells recognizing LPGAP are expanded after infection with L. donovani in humans.

Melatonin concentration as a marker of the circadian phase in patients with obstructive sleep apnoea

OBJECTIVE The effects of obstructive sleep apnoea (OSA) on the markers of glucose metabolism and other hormones are of interest, particularly since there is growing evidence that OSA may be a risk factor for disorders such as insulin resistance. However, interpreting these studies depends on the target hormone not having a diurnal rhythm and the circadian rhythm not being altered by the sleep fragmentation that occurs in OSA. Therefore, the aim of our study was to test the hypothesis that OSA displaces the circadian rhythm. METHODS We carried out a prospective, observational, controlled, parallel study in 22 OSA patients (mean [SD] age: 45.1 [8.8]years; apnoea/hypopnoea index (AHI): 37 [24] events/h) and 22 age matched healthy subjects (age: 47.9 [7.9]years; AHI: 3 [1] events/h). Saliva samples for the measurement of melatonin were collected from participants resting in dim light at 30 min intervals between 19:30 and 22:30 h. Dim light melatonin onset (DLMO), a marker of the circadian phase, was taken at the end of the 30 min interval in which the greatest rise in melatonin occurred. RESULTS The group median (interquartile range) DLMO did not differ in OSA patients compared to healthy subjects (OSA patients: 90 [60-150]min; healthy subjects: 135 [90-150]min, p=0.19). CONCLUSION The circadian phase is the same in OSA patients and healthy subjects using salivary melatonin concentration as a marker of the circadian phase.

Dichotomy in the human CD4+ T‐cell response to Leishmania parasites

Leishmania parasites cause human diseases ranging from self‐healing cutaneous ulcers to fatal systemic infections. In addition, many individuals become infected without developing disease. In mice the two subsets of CD4+ T cells, Thl and Th2, have different effects on the outcome of experimental Leishmania infections. Thl cells producing interferon‐y (IFN‐y) mediate resistance, whereas Th2 cells producing interleukin‐4 (IL‐4) and IL‐10 are associated with susceptibility and exacerbation. Evidence is accumulating that a Th1/Th2 dichotomy in the T‐cell response to Leishmania exists also in humans, and that the balance between subsets of parasite‐specific T cells may play an important regulatory role in determining the outcome of the infections.