Arend H. J. Kolk

A simple and economical direct agglutination test for serodiagnosis and sero-epidemiological studies of visceral leishmaniasis

A simple and economical direct agglutination test for the detection of visceral leishmaniasis is described. Trypsin-treated, Coomassie Brilliant Blue-stained, formalin-preserved promastigotes were used as antigen in re-usable V-well microtitre plates. In 21 patients with recent kala-azar, titres of 1:51200 or higher were found. Cured kala-azar patients treated 4 to 14 months before testing, showed titres in the range of 1:3,200 to greater than 1:51,200. Healthy and diseased controls had titres below 1:1,600 with the exception of African trypanosomiasis patients who showed titres of 1:200 to 1:12,800, overlapping with the titres of cured kala-azar patients. Where trypanosomiasis is not a consideration, a titre of 1:1,600 could be considered indicative of visceral leishmaniasis, the sensitivity and specificity were then 100%. The test was applied to sera of 280 inhabitants of Baringo District, a known focus of visceral leishmaniasis in Kenya. When treated cases were included, the test showed a sensitivity of 100% and specificity of 99.3%. This test could be used in district hospitals and health centres in endemic areas as an aid in diagnosis of kala-azar and in the field for sero-epidemiological studies.

Interleukin-1 Signaling Is Essential for Host Defense during Murine Pulmonary Tuberculosis

Interleukin (IL)-1 signaling is required for the containment of infections with intracellular microorganisms, such as Listeria monocytogenes and Leishmania major. To determine the role of IL-1 in the host response to tuberculosis, we infected IL-1 type I receptor-deficient (IL-1R(-/-)) mice, in which IL-1 does not exert effects, with Mycobacterium tuberculosis. IL-1R(-/-) mice were more susceptible to pulmonary tuberculosis, as reflected by an increased mortality and an enhanced mycobacterial outgrowth in lungs and distant organs, which was associated with defective granuloma formation, containing fewer macrophages and fewer lymphocytes, whereas granulocytes were abundant. Lymphocytes were predominantly confined to perivascular areas, suggesting a defective migration of cells into inflamed tissue in the absence of IL-1 signaling. Impaired host defense in IL-1R(-/-) mice was further characterized by a decrease in the ability of splenocytes to produce interferon-gamma. Analysis of these data suggests that IL-1 plays an important role in the immune response to M. tuberculosis.

Prospects for Clinical Application of Electronic-Nose Technology to Early Detection of Mycobacterium tuberculosis in Culture and Sputum

ABSTRACT Ziehl-Neelsen (ZN) staining for the diagnosis of tuberculosis (TB) is time-consuming and operator dependent and lacks sensitivity. A new method is urgently needed. We investigated the potential of an electronic nose (EN) (gas sensor array) comprising 14 conducting polymers to detect different Mycobacterium spp. and Pseudomonas aeruginosa in the headspaces of cultures, spiked sputa, and sputum samples from 330 culture-proven and human immunodeficiency virus-tested TB and non-TB patients. The data were analyzed using principal-component analysis, discriminant function analysis, and artificial neural networks. The EN differentiated between different Mycobacterium spp. and between mycobacteria and other lung pathogens both in culture and in spiked sputum samples. The detection limit in culture and spiked sputa was found to be 1 × 104 mycobacteria ml−1. After training of the neural network with 196 sputum samples, 134 samples (55 M. tuberculosis culture-positive samples and 79 culture-negative samples) were used to challenge the model. The EN correctly predicted 89% of culture-positive patients; the six false negatives were the four ZN-negative and two ZN-positive patients. The specificity and sensitivity of the described method were 91% and 89%, respectively, compared to culture. At present, the reasons for the false negatives and false positives are unknown, but they could well be due to the nonoptimized system used here. This study has shown the ability of an electronic nose to detect M. tuberculosis in clinical specimens and opens the way to making this method a rapid and automated system for the early diagnosis of respiratory infections.

CpG Oligodeoxynucleotides Enhance Host Defense during Murine Tuberculosis

ABSTRACT Oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs activate immune cells to produce cytokines. CpG ODNs protect mice against infections with intracellular bacteria by the induction of a T helper 1 (Th1) response. To determine the effect of CpG ODNs in pulmonary tuberculosis, mice were treated with CpG ODNs or control ODNs at the time of intranasal infection. CpG ODNs reduced mycobacterial outgrowth for up to 5 weeks after Mycobacterium tuberculosis infection and were associated with a decrease in inflammation in lung tissue. CpG treatment was also associated with elevated levels of gamma interferon (IFN-γ) and decreased levels of interleukin 4 in the lungs and an increased capacity of splenocytes to secrete Th1-type cytokines. CpG ODNs given 2 weeks after infection were still able to reduce mycobacterial outgrowth and to enhance a Th1 response 5 weeks postinfection. Administration of CpG ODNs to IFN-γ-gene-deficient mice failed to reduce mycobacterial outgrowth. These data suggest that CpG ODNs improve host defense during pulmonary tuberculosis by an IFN-γ-dependent mechanism.

Evaluation of a newly developed direct agglutination test (DAT) for serodiagnosis and sero-epidemiological studies of visceral leishmaniasis: comparison with IFAT and ELISA

A newly developed direct agglutination test (DAT) for visceral leishmaniasis, IFAT and ELISA were applied to sera of patients with visceral leishmaniasis, African and American trypanosomiasis, other parasitic infections and healthy controls. The sensitivities of the 3 tests were comparable (96.3% to 100%); excluding patients with African and American trypanosomiasis, the specificities of DAT and IFAT were 100% and ELISA 87.3%. When trypanosomiasis sera were included, the specificities were 72.6%, 94.3% and 79.4% in DAT, IFAT and ELISA respectively. In 273 sera from a leishmaniasis endemic area (Baringo District, Kenya), the sensitivity was 80% in DAT and IFAT and 60% in ELISA, specificities being 99.6% (DAT), 98.5% (IFAT) and 62.5% (ELISA). As the new DAT is economical and easy to perform, it is recommended for sero-epidemiological field work on visceral leishmaniasis.

Early diagnosis of tuberculous meningitis by polymerase chain reaction

Background: Rapid detection of Mycobacterium tuberculosis is of vital importance for patients with tuberculous meningitis. We evaluated an improved polymerase chain reaction (PCR) technique for rapid and specific identification of M tuberculosis in CSF. Methods: The technique was used on CSF samples from 42 patients (3 of whom were human immunodeficiency virus seropositive) with clinical symptoms, signs, and laboratory findings suggestive of tuberculous meningitis. The target for amplification was a nucleotide sequence located within IS6110. A small amount of DNA from M smegmatis strain 1008, containing a modified IS6110, was added in the PCR as a control for inhibitors and to quantitate the PCR for M tuberculosis. Results: On the basis of symptoms and clinical findings, antituberculous treatment was started in 35 patients, but was later stopped in 11 because of lack of response. From 12 patients responding to treatment and with a positive diagnostic test, 11 cases were detected by PCR, nine cases were culture positive, and two cases microscopy positive. Of the remaining 12 patients who had negative CSF by microscopy, PCR, and culture, 11 were diagnosed as having tuberculous meningitis on the basis of the response to treatment (three had active pulmonary tuberculosis) and one had mycobacteria other than M tuberculosis in sputum and urine. The sensitivity of the PCR was 48% in those with a final diagnosis of tuberculous meningitis (culture or PCR confirmed cases, plus those with clinical evidence and who responded to antituberculous treatment), which is much higher than the 9% sensitivity of microscopy. There were no false-positive PCR results. Conclusions: PCR on CSF is a rapid method for the accurate diagnosis of tuberculous meningitis.

Measurement of antigen specific lymphocyte proliferation using 5-bromo-deoxyuridine incorporation An easy and low cost alternative to radioactive thymidine incorporation

The classical in vitro assay for the determination of cell mediated immune responses is the lymphocyte transformation test (LTT) in which cell proliferation is measured by incorporation of radioactive labeled thymidine (3H-TdR). The LTT assay using 3H-TdR is less suited for modestly equipped laboratories as it is costly, laborious and involves the need to handle radioactive isotopes and specialized equipment. Here we describe an improved alternative LTT method which is capable of detecting specific cellular immune reactions (CMI) against (mycobacterial) antigens in vitro. This assay, the bromodeoxyuridine-ELISA LTT test, is simple, less expensive, reproducible and is as sensitive as the 3H-TdR test. The specific advantages of the test are a simple denaturation step and the fact that no radioactive isotopes are needed. The test is specifically suited for research laboratories in tropical countries which study CMI in those human infectious diseases where this arm of the immune response plays a pivotal role in the generation of immunity, e.g., in tuberculosis, leprosy and leishmaniasis.

Molecular and immunological analysis of a fibronectin‐binding protein antigen secreted by Mycobacterium leprae

By screening a Mycobacterium lepraeλgt11 genomic DNA library with leprosy‐patient sera we have previously identified 50 recombinant clones that expressed novel M. leprae antigens (Sathish et al., 1990). In this study, we show by DNA sequencing and immunoblot analysis that three of these clones express a M. leprae homologue of the fibronectin‐binding antigen 85 complex of mycobacteria. The complete gene was characterized and it encodes a 327‐amino‐acid polypeptide, consisting of a consensus signal sequence of 38 amino acids followed by a mature protein of 289 amino acids. This is the first sequence of a member of the M. leprae antigen 85 complex, and Southern blotting analysis indicated the presence of multiple genes of the 85 complex in the genome of M. leprae. The amino acid sequence displays 75–85% sequence identity with components of the antigen 85 complex from M. tuberculosis, M. bovis BCG and M. kansasii. Furthermore, antibodies to the antigen 85 complex of M. tuberculosis and M. bovis BCG reacted with two fusion proteins containing the amino acid regions 55–266 and 265–327 of the M. leprae protein. The M. leprae 30/31 kDa protein induces strong humoral and cellular responses, as judged by Western blot analysis with patient sera and proliferation of T cells derived from healthy individuals and leprosy patients. Amino acid regions 55–266 and 265–327 both were shown to bind to fibronectin, indicating the presence of at least two fibronectin‐binding sites on the M. leprae protein. These data indicate that this 30/31 kDa protein is not only important in the immune response against M. leprae, but may also have a biological role in the interaction of this bacillus with the human host.

Antigens in culture supernatant of Mycobacterium tuberculosis: epitopes defined by monoclonal and human antibodies

Antigens of Mycobacterium tuberculosis found in the supernatant of heat-treated cultures were characterized in order to explore whether antigens from this source could be used for the development of a serological test. Culture supernatants and sonicates of 12, 25 and 39 d cultures were analysed by SDS-PAGE. In culture supernatant, major protein bands of 65, 24, and 12 kDa were visible after Coomassie brilliant blue staining. Using murine monoclonal antibodies in Western blots, a pattern of protein bands distinct from that of the corresponding M. tuberculosis sonicates was found in all the culture supernatants. Gel permeation chromatography, in the presence of SDS, was used to separate the major protein bands in the culture supernatant. In ELISA, sera from 20 of 26 patients with tuberculosis reacted with fractions containing mainly 24 kDa or 12 kDa proteins, whereas none of the control sera reacted. In Western blots, each patient serum had its own characteristic banding pattern with culture supernatant, but all the sera from tuberculosis patients and control subjects reacted with protein bands of 65, 61, 58, 30 and 24 kDa. The 12 kDa protein was recognized only by sera from patients with tuberculosis in both Western blots and ELISA. This suggests that different kinds of epitopes on proteins of M. tuberculosis are detected by human antibodies in Western blots and ELISA. We assume that epitopes recognized in Western blots by patients with tuberculosis and control subjects are ubiquitous and are also present on normal commensal bacteria. Epitopes recognized by only some patients with tuberculosis in Western blots may be linear and M. tuberculosis specific. Epitopes recognized by tuberculosis patients but by none of the control subjects in ELISA may be conformation related and M. tuberculosis specific. The major protein bands found in supernatants of heat-treated cultures, 24 and 12 kDa, possess epitopes that may be M. tuberculosis specific and are potentially valuable for the development of a serological test.

Electronic-Nose Technology Using Sputum Samples in Diagnosis of Patients with Tuberculosis

ABSTRACT We investigated the potential of two different electronic noses (EN; code named “Rob” and “Walter”) to differentiate between sputum headspace samples from tuberculosis (TB) patients and non-TB patients. Only samples from Ziehl-Neelsen stain (ZN)- and Mycobacterium tuberculosis culture-positive (TBPOS) sputum samples and ZN- and culture-negative (TBNEG) samples were used for headspace analysis; with EN Rob, we used 284 samples from TB suspects (56 TBPOS and 228 TBNEG samples), and with EN Walter, we used 323 samples from TB suspects (80 TBPOS and 243 TBNEG samples). The best results were obtained using advanced data extraction and linear discriminant function analysis, resulting in a sensitivity of 68%, a specificity of 69%, and an accuracy of 69% for EN Rob; for EN Walter, the results were 75%, 67%, and 69%, respectively. Further research is still required to improve the sensitivity and specificity by choosing more selective sensors and type of sampling technique.