Challenges of tuberculosis prevention through early detection of latent tuberculosis infection in new immigrants to the State of Kuwait

Introduction: Despite management advances worldwide, tuberculosis still remains a serious uncontrolled disease. The absence of either a ‘gold’ standard diagnostic test, or a conventional rapid ‘reference’ laboratory test for asymptomatic Mycobacterium tuberculosis (MTB) carriers complicates disease control. Through mandatory screening of high-risk groups, early diagnosis of latent tuberculosis infection (LTBI) cases allows recognition and better control of the tuberculosis pandemic. Materials and Methods: The current tuberculosis screening guidelines as recommended by the World Health Organization, chest X-ray and tuberculin skin test were assessed and revealed rises in TB morbidity and fatality trends in the Kuwait population (low incidence country). In order to evaluate options for LTBI diagnosis, the current work implemented a 4-month prospective, observational, repeated-measure and randomly implemented survey on 180 new immigrants to Kuwait using a structured risk factor questionnaire whilst, simultaneously evaluating the performance of the two standard diagnostics (chest X-ray and tuberculin skin test) with the new biomarker interferon gamma release assays (T-SPOT .TB test and QuantiFERON Gold In-Tube test (QNF-GIT)); which detect the release of interferon gamma (INF-γ) released from sensitization to specific MTB antigens. Results: Associations between various epidemiological risk factors - such as socio-demographic status, smoking and environmental exposure-contact - were associated in the laboratory diagnosed LTBI participants. Positive identification of LTBI prevalence detected by two radiologists was 10.1% having ‘moderate’ inter-reader agreement (Kappa = 0.505), compared to no positives being detected by three pulmonologists. TST results were negative (less than 10-mm ‘cut-off’) even in the 86.1% Bacillus Calmette-Guérin vaccinated expatriates. Estimated LTBI using QNFGIT was 28.3% compared to 41.1% positive T-SPOT .TB test. Both interferon gamma assays revealed concordant ‘abnormal’ results in 26.1% with ‘good’ agreement (kappa = 0.627). Conclusion: Detection of latent tuberculosis infection can be facilitated by introducing evidence-based diagnostic classification depending on history taking of epidemiological-related risk factors and chest X-ray plus either interferon gamma assays

Impact of Helminth Infection on Antimycobacterial Immune Responses in UK Migrants

Tuberculosis and helminth infections are co endemic in many parts of the world. This geographical overlap has led to the hypothesis that helminth infections could exacerbate the effects of Mycobacterium tuberculosis (Mtb) infection. Anthelmintic treatment has been observed to be associated with improved mycobacterial cellular responses and decreases in the frequency of Treg cells. The consequence of this immunomodulation may affect the ability of the host to restrict the growth of mycobacteria or mycobacterial killing. This study aims at investigating the modulations of the immune response profile of latent tuberculosis (LTBI) and helminth co-infected patients and whether these modulations are associated with a decrease in mycobacterial growth inhibition using a mycobacterial growth inhibition assay (MGIA).UK migrants attending University College Hospital London, UK with eosinophilia or suspected/diagnosed helminth infection (Strongyloides spp and Schistosoma spp) and/or LTBI were bled at recruitment (before anthelmintic treatment) and 4 months after completing anthelmintic treatment. Helminth infected patients displayed poor growth inhibition on MGIA which was improved after anthelmintic treatment which indicated this immunomodulation might be helminth mediated. The percentage of CD4+ T cells expressing IFNg, TNFa and IL-2 were quantified by flow cytometry in PPD and ESAT-6/CFP-10 stimulated PBMC and anthelmintic treatment was observed to increase the frequency of CD4+IFNg response. LTBI-helminth coinfection was associated with significantly elevated levels of proinflammatory and lower levels of anti-inflammatory cytokines after they were treated. IP- 10 was significantly upregulated and MCP-1 was significantly downregulated in LTBIhelminth coinfected patients after anthelmintic treatment. The effect of IL-10 and TGFb on MGIA were observed and suggested an immunoregulatory role in helminth infected patients. Gene expression analysis by qRT-PCR showed varied responses and showed significant fold changes of CXCL-10, arginase 1 and CD163 after the treatment. MGIA and multiple immune parameters have shown that helminth infection can modulate a variety of Mtb specific immune responses

Tuberculosis

Data are rapidly accumulating from all over the world regarding the efficacy of standardized treatment regimens for drug-sensitive, drug-resistant TB and latent TB infection. While we are facing the menace of multi drug-resistant TB [MDR-TB], extensively drug-resistant tuberculosis [XDR­ TB] has emerged threatening to undermine global efforts at TB control. Hence we have included chapters to cover all aspects of the diagnosis and management of MDR TB. This book will cover all these developments in great detail. With the widespread availability of internet globally various standard web resources available on TB have also been included so that the readers may get the comprehensive and updated guidelines from these resources. The changing clinical presentation of TB, advances in laboratory, imaging diagnostic modalities, therapeutic measures and emergence of MDR TB all suggest a pressing need to have a updated book on TB. Furthermore, while all physicians encounter the TB disease in their clinical practice, there have been a lot of controversies and misconceptions over various issues for the diagnosis and management of TB. Paucity of a well referenced, updated, standard book of TB has prompted us to undertake this venture sharing the clinical experience of global experts of TB. Our book contains chapters on epidemiology, immune-pathology, diagnosis, treatment and latest advances for TB, highlighting the global perspective of tuberculosis. World-wide resurgence of MDR TB indicates that the battle against this foe of mankind will continue in the coming years. TB still remains to be a research priority of paramount importance from medical, social and financial aspects and we have attempted to highlight all the aspects for the treatment of TB. We believe that this book will serve as a practical guide for the diagnosis and management of TB for practicing physicians (especially pulmonologists and internists) and all those who are involved in the management of TB

Influence of Genetic Polymorphism Towards Pulmonary Tuberculosis Susceptibility

Tuberculosis (TB) is still remains the major threat for human health worldwide. Several case-control, candidate-gene, family studies and genome-wide association studies (GWAS) suggested the association of host genetic factors to TB susceptibility or resistance in various ethnic populations. Moreover, these factors modulate the host immune responses to tuberculosis. Studies have reported genetic markers to predict TB development in human leukocyte antigen (HLA) and non-HLA genes like killer immunoglobulin-like receptor (KIR), toll-like receptors (TLRs), cytokine/chemokines and their receptors, vitamin D receptor (VDR) and SLC11A1 etc. Highly polymorphic HLA loci may influence antigen presentation specificities by modifying peptide binding motifs. The recent meta-analysis studies revealed the association of several HLA alleles in particular class II HLA-DRB1 with TB susceptibility and valuable marker for disease development especially in Asian populations. Case-control studies have found the association of HLA-DR2 in some populations, but not in other populations, this could be due to an ethnic specific association of gene variants. Recently, GWAS conducted in case-control and family based studies in Russia, Chinese Han, Morocco, Uganda and Tanzania revealed the association of genes such as ASAP1, Alkylglycerol monooxygenase (AGMO), Forkhead BoxP1 (FOXP1), C-terminal domain phosphatase 1 (UBLCP1) and intergenic SNP rs932347C/T with TB. Whereas, SNP rs10956514A/G were not associated with TB in western Chinese Han and Tibetan population. In this review, we summarize the recent findings of genetic variants with susceptibility/resistance to TB

Tuberculosis– meeting the challenge of a global pandemic at molecular level

We showed that a well established epidemic in rural Bangladesh represented a low rate of recent transmission. A majority of the strains belonged to the ancient East African- Indian (EAI) lineage (Paper I). Our study found that the total resistance among new cases to any drug was 31%, 2% were MDR. The NTP in Sunamganj is still effective, although the high resistance to INH is alarming (Paper II), and DST services were possible and highly needed. Due to the low rate of transmission in the high-incidence area, it was likely that most patients had been infected a long time ago. We thus wanted to measure the impact DD could have in similar high incidence settings (Paper IV). In order to study the impact of DD, an introductory review of published studies describing DD in numerous countries was conducted (Paper III). We found the core problem in DD to be a vicious cycle of repeated visits at the same healthcare level, resulting in non-specific antibiotic treatment and failure to access specialized TB services. To reduce the DD there is an urgent need for alternative means to monitor the epidemic at the local level. We found that a systematic registration of treatment delay in the TB program records of the Amhara Region of Ethiopia could be utilized to estimate the infectious pool of TB. By recording the treatment delay for new TB cases, retreatment cases and failures, and by estimating the number of undiagnosed cases, the total number of infectious days and, hence, an estimate of the infectious pool could be calculated (Paper IV). Since DD was considerable in the high-incidence setting we wanted to compare to risk-groups in developed countries with a low-rate of TB (Paper V). Health care workers exposed to TB at three Norwegian hospitals as well as a non-exposed control group were tested with both TST and the INF-ã test T-SPOT.TB. Our data indicate that the frequency of latent TB in the total cohort of HCWs is 3% whereas the rate of transmission of TB to exposed individuals is around 2% and occurs through short time exposure. Thus, the risk of TB transmission to health care workers following unprotected TB exposure in a hospital setting in Norway is low

Clinical, Immunological and Therapeutic Aspects of Lymphatic Filariasis

Lymphatic filariasis is a major health problem in many countries of the world. The disease still takes a heavy toll in terms of social and economic losses. • The commonest clinical forms seen in a large clinic in an endemic area are lymphoedema of the lower limbs, hydrocoele, lymphoedema of the genitalia, chyluria and tropical eosinophilia. • Apart from filarial oedema, the most important clinical manifestation of filariasis is the episodes of adenolymphangitis. The brief duration and episodic nature of ADL makes it the best manifestation of lymphatic filarial disease that can be well studied. • These ADL episodes contribute significantly to the morbidity associated with the disease. Recurrent attacks of lymphangitis lead on to the progression of lymphoedema and development of elephantiasis. • Patients with higher grades of lymphoedema were more predisposed to ADL attacks. Thus, while there were ADL episodes in patients with Grade III oedema, they were only in grade II and in Grade I. This can be explained by the predisposition to fungal infection in limbs with higher grades of oedema. • This study has shown that any factor resulting in injury to the skin of the oedematous limb promotes secondary bacterial infection, which is the most important cause for the ADL episodes. Fungal infection of the affected limb, especially on constant exposure to moisture, is the commonest predisposing factor, followed by minor injuries and infections. • As in the case of the analysis done according to drug groups there was an increase in the number of ADL attacks in the follow up phase when compared to the treatment year. A response in the form of reduction in the episodes of ADL in the treatment groups; oral penicillin group alone, DEC group alone and the combination; as well as the foot care group was significant (p<0.008). This was however statistically significant only for patient with grade III (p<0.025) and grade IV (p<0.009) oedema. • The reductions were greater in the lower grades of oedema as compared with the higher grades although the difference between the groups were not statistically significant. There was an increase in the number of attacks in the follow up year. • The importance of the role of bacteria entering through breaks in the skin and precipitating ADL attacks has been convincingly demonstrated. • Further long-termstudies, which would help to document that the lymphoedema of early filarial oedema can be reversed by using antibiotics and carefully designed limb care programmes. This beneficial effect would help to enhance the success of large-scale control programmes since the perceived benefit by the affected persons would be high. • Advanced Multi modality treatment for the reversal of grade IV lemphedema patient has been performed with nodovenal shunt in order to get best outcome in cosmetic and functional limb. • Helminth infections in the study area were highly prevalent. Geohelminths and filarial infections were seen maximally in the younger years of life. • PPD sensitivity was influenced by the presence of helminth infections. • Clearance of helminth infections may provide better response to a variety of vaccines. • Targeting of these pathways, as well as new treatment regimens involving TLR modulation, could potentially provide new avenues in ameliorating pathology in chronic lymphatic filariasis. In addition, our data also reveal a new link between innate immune responses (in the form of TLR activation) and angiogenic/lymphangiogenic pathways (in the form of elevated growth factors), thereby providing additional insight into the pathways connecting the immune system and normal physiologic processes such as lymphatic circulation. • Alteration of TLR expression and function in filaria- infected individuals with latent TB can have major implications in the control of latent TB infection. In addition, these findings also have significant implications for vaccine efficacy in helminth- endemic countries. Vaccines requiring a pro-inflammatory cytokine response for efficacy and those involving TLR agonists as adjuvants may not function optimally in the presence of helminth coinfection. The reversal of TLR modulation upon treatment of filarial infection suggests that elimination of this helminth infection in endemic areas might have a profound effect in the control of TB infection

The validity and limitations of IGRA (QFT-GIT) and TST in screening for pulmonary tuberculosis among adults applying for residency in the Emirate of Abu Dhabi, UAE in 2013

Mycobacterium tuberculosis (MTB), the agent causing tuberculosis (TB) and latent tuberculosis infection (LTBI) imposes a significant public health concern world-wide. The utility and the value for simultaneous (parallel) testing with Interferon Gamma Release Assay [IGRA (QFT-GIT)] and Tuberculin skin test (TST) in detecting MTB infection among applicants for residency in Abu Dhabi requires evaluation. The objectives of this study are to 1) Provide demographic characteristics of the study sample by age categories, and 2) Explore the validity measures of IGRA (QFT-GIT) and TST, and 3) Further, to assess the risk of MTB among younger visa applicants compared to older applicants. This cross-sectional study analyzes data routinely collected on all adults (18-64 years) seeking a residency visa in the Emirate of Abu Dhabi from January – December 2013. Among the 1,529,389 adults who applied for this visa in 2013, 2,596 individuals presented with an abnormal chest X-ray (and who also were HIV negative). In our study, nearly two-thirds of males in the sample were from Pakistan and India while almost 50% of females were from the Philippines. The sensitivity of IGRA (QFT-GIT) test was 72.7%, (95% CI: 67.8-77.2) compared to TST sensitivity of 59.1%, (95% CI: 53.8-64.2), (P<0.001). The net sensitivity of simultaneous (parallel) testing was 88.8%. The net sensitivity of simultaneous (parallel) testing for the younger age group (18-34) years was 91.6% compared to 83.1% net sensitivity among older individuals (35-64) years. Kappa agreement between IGRA (QFT-GIT) and TST was 0.33 (95% CI: 0.29-0.36). The role of chest X-ray in our study was not adequate and older individuals (35-64) years had lower proportions of MTB infection when compared to younger individuals (18-34) years, yielding an Odds Ratio (OR) of 0.62 (95% CI 0.48– 0.81), (P<0.001). In conclusion, nearly two-thirds of males in the sample were from Pakistan and India. IGRA (QFT-GIT) was more sensitive than TST. The net sensitivity of simultaneous (parallel) testing was high. However, Kappa agreement between IGRA (QFT-GIT) and TST in the study was considered as only fair concordance. Primary Reader: David Celentano Secondary Readers: Lilly Engineer, Jonathan Golub, Josh Sharfstein, Amal Mohamme

Molecular epidemiology and phenotypic and genetic characterization of <i>Mycobacteria</i> isolated in central Vietnam

The overall objective of this study is to determine molecular epidemiology and phenotypic and genetic characterization of mycobacteria isolated in central Vietnam. In this study, the MODS assay was used to isolate mycobacteria and determine the proportion of drug resistant strains in 252 clinical samples. M.tuberculosis was detected in 153 samples (60.7%) and 46 (30.1%) were antibiotic resistant. One drug resistance was present in 30 strains (19.6%): 18 for RIF, 6 for INH and 3 for STR and EBM. Multidrug resistant M. tuberculosis as defined by WHO (resistant to RIF and INH) was observed in 13 strains. There were additional 14 strains showing resistance to two or more drugs. Identification of M. tuberculosis using the 16S rDNA based PCR assay detected 258 positive in 480 clinical samples(53.8%), including 73 smear negative cases. The IS6110 PCR of positive cases could not confirm the positivity in 3 samples, probably due to the absence of the insertion sequence. Spoligotyping produced a total of 36 different patterns for the 122 M. tuberculosis strains examined. The EAI family genotype (65.6% of isolates) dominated in our study: 15.6 % were of the EAI4-VNM genotype and 46.7 % were of EAI5. Beijing genotype was observed in 12.3 % of isolates. There were 4 isolates (3.3%) in which spoligotype pattern did not match with any of the updated international spoligotype database of the M. tuberculosis complex -SpolDB4. The remaining 22% were of other genotypes (including U, T, MANU, H and CAS). Sequence and analysis of the whole genome of MTB_HUE_20 strain - a multidrug resistant strain with unknown spoligotype pattern - showed that it is 4,397,928 bp in length and contains 64.8% of GC. This strain harbors only one copy of IS6110 and has a mutation on katG gene

The antimycobacterial activity of thioridazine derivatives against drug resistant Mycobacterium tuberculosis: in vitro, ex vivo and in vivo studies

O objectivo principal desta tese foi o de avaliar a acção da tioridazina (TZ), bem como de compostos derivados desta, obtidos por manipulação química, como agentes com actividade contra Mycobacterium tuberculosis, em particular contra M. tuberculosis multi-resistente (MDR-TB). Desta forma foram obtidos vinte e dois derivados e efectuados testes de toxicidade e mutagenicidade pelo método de exclusão com azul de trypan (realizado em linfócitos humanos) e o teste de Ames, respectivamente. Todos os derivados não tóxicos e não mutagénicos foram testados in vitro contra estirpes de Staphylococcus aureus resistente à meticilina (MRSA), que foi o microrganismo modelo utilizado durante todo o trabalho, e estirpes de MDR-TB. Visto que a tuberculose é uma infecção do macrófago alveolar, estes estudos foram subsequentemente aplicados a macrófagos infectados. Desta forma, é importante analisar a actividade que estes compostos apresentam dentro do macrófago, local onde normalmente a micobactéria se encontra. Iniciaram-se estudos animais com a TZ, de forma a verificar a eficácia deste composto em curar murganhos Balb/C infectados com M. tuberculosis H37Rv ATCC27294. Desta forma foi possível optimizar parâmetros, tais como, a via de infecção, a concentração de composto a administrar, entre outros. Os resultados obtidos demonstraram que dos vinte e dois derivados nenhum apresentava toxicidade ou efeitos mutagénicos, nas condições testadas. Desta forma, os vinte e dois derivados foram seleccionados para estudos in vitro contra estirpes de S. aureus e de M. tuberculosis. Dos estudos in vitro foi possível verificar que seis derivados apresentaram uma maior actividade do que a TZ e desta forma foram seleccionados para os estudos ex vivo. Quando testados em macrófagos infectados três derivados demonstraram um efeito marcado na activação das células fagocitárias (“enhancement of the killing activity”), sendo um dos derivados ainda mais activo do que a TZ. Dos estudos em animais, foi possível seleccionar as condições a serem implementadas em estudos futuros com os derivados mais activos. De todos os resultados obtidos durante esta tese foi possível desenvolver um modelo baseado na interacção do macrófago com a bactéria e a subsequente acção destes compostos. O modelo desenvolvido (“macrophage model”) pode assim contribuir para clarificar o que ocorre a nível intracelular aquando da adição dos compostos ao meio de cultura.The main objective of this Thesis was to evaluate thioridazine (TZ) and chemically derived derivatives for anti-Mycobacterium tuberculosis activity in particular against multi-drug resistant (MDR) M. tuberculosis. Twenty-two TZ derivatives were obtained and screened for toxicity and mutagenicity by the trypan blue exclusion assay in human lymphocytes and the Ames test, respectively. The derivatives that were devoid of any toxicity and mutagenicity were then tested against Methicillin-resistant Staphylococcus aureus (MRSA) used as a model during the entire work, and against antibiotic resistant M. tuberculosis (MDR-TB) strains in vitro and subsequently in the macrophage that has phagocytosed the organism. Since tuberculosis is an infection of the alveolar macrophage it is important to evaluate the activity of these compounds inside the phagocytic cell where the mycobacteria are to be found. Thioridazine was also tested for its ability to cure the mouse of infection by M. tuberculosis. Therefore, animal studies using Balb/C mice infected with M. tuberculosis H37Rv ATCC27294 strain were initiated to parameterize the route of infection, dose of compound to be administered, among others. The results obtained showed that from the twenty-two TZ derivates none was toxic or mutagenic under the conditions tested. Therefore, all the twenty-two derivatives were selected for in vitro evaluation against S. aureus and M. tuberculosis strains. From the in vitro results six derivatives showed greater activity than TZ and were selected for ex vivo studies. Three of these derivatives were shown to enhance the macrophage killing activity and one of the derivatives was even more active than TZ. From the animal studies it was possible to select the conditions to apply in further studies with the most active derivatives. From all the data obtained during this Thesis it was possible to develop a model based on the macrophage interaction with the bacteria and the subsequent action of the compounds. The macrophage model can elucidate what takes place inside the human macrophage when these compounds are added to the medium. Due to the uniqueness of the approaches developed during the Thesis research, other potential sources of anti-tubercular compounds were explored: plants and organosilicon compounds (SILA). These studies demonstrated that extracts from the nuisance plant Carpobrotus edulis could enhance the killing of intracellular bacteria such as MDR-TB and MRSA. In addition, the extract was shown to modulate the immune system thereby indicating its potential use for cellular immune deficient disorder as well as render MDR mouse lymphoma cells carrying the human mdr1 gene completely susceptible to cytotoxic drugs to which they were initially resistant. The results of this component of my research have resulted in the design of new experiments which are now being carried out by another Ph.D. student in our Unit and at the Medical University of Szeged, Hungary. In conclusion, the results obtrained from my Thesis research has paved the way for clinical trial consideration of the many compounds studied shown to have significant intracellular activity against XDR-TB/MDR-TB at concentrations that are non-toxic and that can be readily achieved in the infected human