Serial measurement of the circulating levels of tumour necrosis factor and its soluble receptors 1 and 2 for monitoring leprosy patients during multidrug treatment

Leprosy is an infectious and contagious spectral disease accompanied by a series of immunological events triggered by the host response to the aetiologic agent, Mycobacterium leprae . The induction and maintenance of the immune/inflammatory response in leprosy are linked to multiple cell interactions and soluble factors, primarily through the action of cytokines. The purpose of the present study was to evaluate the serum levels of tumour necrosis factor (TNF)-α and its soluble receptors (sTNF-R1 and sTNF-R2) in leprosy patients at different stages of multidrug treatment (MDT) in comparison with non-infected individuals and to determine their role as putative biomarkers of the severity of leprosy or the treatment response. ELISA was used to measure the levels of these molecules in 30 healthy controls and 37 leprosy patients at the time of diagnosis and during and after MDT. Our results showed increases in the serum levels of TNF-α and sTNF-R2 in infected individuals in comparison with controls. The levels of TNF-α, but not sTNF-R2, decreased with treatment. The current results corroborate previous reports of elevated serum levels of TNF-α in leprosy and suggest a role for sTNF-R2 in the control of this cytokine during MDT.

Incidence and transmission patterns of tuberculosis among indigenous populations in Brazil

Approximately 10% of the Brazilian indigenous population lives in the state of Mato Grosso do Sul (MS), where a large number of new cases of tuberculosis (TB) are reported. This study was conducted to assess TB occurrence, transmission and the utility of TB diagnosis based on the Ogawa-Kudoh (O-K) culture method in this remote population. The incidence of TB was estimated by a retrospective review of the surveillance data maintained by the Notifiable Diseases Surveillance System for the study region. The TB transmission pattern among indigenous people was assessed by genotyping Mycobacterium tuberculosis isolates using the IS 6110restriction fragment length polymorphism (RFLP) technique. Of the 3,093 cases identified from 1999-2001, 610 (~20%) were indigenous patients (average incidence: 377/100,000/year). The use of the O-K culture method increased the number of diagnosed cases by 34.1%. Of the genotyped isolates from 52 indigenous patients, 33 (63.5%) belonged to cluster RFLP patterns, indicating recently transmitted TB. These results demonstrate high, on-going TB transmission rates among the indigenous people of MS and indicate that new efforts are needed to disrupt these current transmissions.

Comparative clinical study of different multiplex real time PCR strategies for the simultaneous differential diagnosis between extrapulmonary tuberculosis and focal complications of brucellosis

Background: Both brucellosis and tuberculosis are chronic-debilitating systemic granulomatous diseases with a high incidence in many countries in Africa, Central and South America, the Middle East and the Indian subcontinent. Certain focal complications of brucellosis and extrapulmonary tuberculosis are very difficult to differentiate clinically, biologically and radiologically. As the conventional microbiological methods for the diagnosis of the two diseases have many limitations, as well as being time-consuming, multiplex real time PCR (M RT-PCR) could be a promising and practical approach to hasten the differential diagnosis and improve prognosis. Methodology/Principal Findings: We designed a SYBR Green single-tube multiplex real-time PCR protocol targeting bcsp31 and the IS711 sequence detecting all pathogenic species and biovars of Brucella genus, the IS6110 sequence detecting Mycobacterium genus, and the intergenic region senX3-regX3 specifically detecting Mycobacterium tuberculosis complex. The diagnostic yield of the M RT-PCR with the three pairs of resultant amplicons was then analyzed in 91 clinical samples corresponding to 30 patients with focal complications of brucellosis, 24 patients with extrapulmonary tuberculosis, and 36 patients (Control Group) with different infectious, autoimmune or neoplastic diseases. Thirty-five patients had vertebral osteomyelitis, 21 subacute or chronic meningitis or meningoencephalitis, 13 liver or splenic abscess, eight orchiepididymitis, seven subacute or chronic arthritis, and the remaining seven samples were from different locations. Of the three pairs of amplicons (senX3-regX3+ bcsp3, senX3-regX3+ IS711 and IS6110+ IS711) only senX3-regX3+ IS711 was 100% specific for both the Brucella genus and M. tuberculosis complex. For all the clinical samples studied, the overall sensitivity, specificity, and positive and negative predictive values of the M RT-PCR assay were 89.1%, 100%, 85.7% and 100%, respectively, with an accuracy of 93.4%, (95% CI, 88.3—96.5%). Conclusions/Significance: In this study, a M RT-PCR strategy with species-specific primers based on senX3-regX3+IS711 sequences proved to be a sensitive and specific test, useful for the highly efficient detection of M. tuberculosis and Brucella spp in very different clinical samples. It thus represents an advance in the differential diagnosis between some forms of extrapulmonary tuberculosis and focal complications of brucellosis.

Insights from paleomicrobiology into the indigenous peoples of pre-colonial America - A Review

This review investigates ancient infectious diseases in the Americas dated to the pre-colonial period and considers what these findings can tell us about the history of the indigenous peoples of the Americas. It gives an overview, but focuses on four microbial pathogens from this period: Helicobacter pylori, Mycobacterium tuberculosis, Trypanosoma cruzi and Coccidioides immitis, which cause stomach ulceration and gastric cancer, tuberculosis, Chagas disease and valley fever, respectively. These pathogens were selected as H. pylori can give insight into ancient human migrations into the Americas, M. tuberculosis is associated with population density and urban development, T. cruzi can elucidate human living conditions and C. immitis can indicate agricultural development. A range of methods are used to diagnose infectious disease in ancient human remains, with DNA analysis by polymerase chain reaction one of the most reliable, provided strict precautions are taken against cross contamination. The review concludes with a brief summary of the changes that took place after European exploration and colonisation.

Examining ERBB2 as a candidate gene for susceptibility to leprosy (Hansen’s disease) in Brazil

Leprosy remains prevalent in Brazil. ErbB2 is a receptor for leprosy bacilli entering Schwann cells, which mediates Mycobacterium leprae-induced demyelination and the ERBB2 gene lies within a leprosy susceptibility locus on chromosome 17q11-q21. To determine whether polymorphisms at the ERBB2 locus contribute to this linkage peak, three haplotype tagging single nucleotide polymorphisms (tag-SNPs) (rs2517956, rs2952156, rs1058808) were genotyped in 72 families (208 cases; 372 individuals) from the state of Pará (PA). All three tag-SNPs were associated with leprosy per se [best SNP rs2517959 odds ratio (OR) = 2.22; 95% confidence interval (CI) 1.37-3.59; p = 0.001]. Lepromatous (LL) (OR = 3.25; 95% CI 1.37-7.70; p = 0.007) and tuberculoid (TT) (OR = 1.79; 95% CI 1.04-3.05; p = 0.034) leprosy both contributed to the association, which is consistent with the previous linkage to chromosome 17q11-q21 in the population from PA and supports the functional role of ErbB2 in disease pathogenesis. To attempt to replicate these findings, six SNPs (rs2517955, rs2517956, rs1810132, rs2952156, rs1801200, rs1058808) were genotyped in a population-based sample of 570 leprosy cases and 370 controls from the state of Rio Grande do Norte (RN) and the results were analysed using logistic regression analysis. However, none of the associations were replicated in the RN sample, whether analysed for leprosy per se, LL leprosy, TT leprosy, erythema nodosum leprosum or reversal reaction conditions. The role of polymorphisms at ERBB2 in controlling susceptibility to leprosy in Brazil therefore remains unclear.

In house reverse membrane hybridisation assay versus GenoType MTBDRplus and their performance to detect mutations in the genes rpoB, katG and inhA

Drug-resistant tuberculosis (TB) threatens global TB control and is a major public health concern in several countries. We therefore developed a multiplex assay (LINE-TB/MDR) that is able to identify the most frequent mutations related to rifampicin (RMP) and isoniazid (INH) resistance. The assay is based on multiplex polymerase chain reaction, membrane hybridisation and colorimetric detection targeting of rpoB and katG genes, as well as the inhA promoter, which are all known to carry specific mutations associated with multidrug-resistant TB (MDR-TB). The assay was validated on a reference panel of 108 M. tuberculosis isolates that were characterised by the proportion method and by DNA sequencing of the targets. When comparing the performance of LINE-TB/MDR with DNA sequencing, the sensitivity, specificity and agreement were 100%, 100% and 100%, respectively, for RMP and 77.6%, 90.6% and 88.9%, respectively, for INH. Using drug sensibility testing as a reference standard, the performance of LINE-TB/MDR regarding sensitivity, specificity and agreement was 100%, 100% and 100% (95%), respectively, for RMP and 77%, 100% and 88.7% (82.2-95.1), respectively, for INH. LINE-TB/MDR was compared with GenoType MTBDRplus for 65 isolates, resulting in an agreement of 93.6% (86.7-97.5) for RIF and 87.4% (84.3-96.2) for INH. LINE-TB/MDR warrants further clinical validation and may be an affordable alternative for MDR-TB diagnosis.

HIV infection disrupts the sympatric host-pathogen relationship in human tuberculosis.

The phylogeographic population structure of Mycobacterium tuberculosis suggests local adaptation to sympatric human populations. We hypothesized that HIV infection, which induces immunodeficiency, will alter the sympatric relationship between M. tuberculosis and its human host. To test this hypothesis, we performed a nine-year nation-wide molecular-epidemiological study of HIV-infected and HIV-negative patients with tuberculosis (TB) between 2000 and 2008 in Switzerland. We analyzed 518 TB patients of whom 112 (21.6%) were HIV-infected and 233 (45.0%) were born in Europe. We found that among European-born TB patients, recent transmission was more likely to occur in sympatric compared to allopatric host-pathogen combinations (adjusted odds ratio [OR] 7.5, 95% confidence interval [95% CI] 1.21-infinity, p = 0.03). HIV infection was significantly associated with TB caused by an allopatric (as opposed to sympatric) M. tuberculosis lineage (OR 7.0, 95% CI 2.5-19.1, p<0.0001). This association remained when adjusting for frequent travelling, contact with foreigners, age, sex, and country of birth (adjusted OR 5.6, 95% CI 1.5-20.8, p = 0.01). Moreover, it became stronger with greater immunosuppression as defined by CD4 T-cell depletion and was not the result of increased social mixing in HIV-infected patients. Our observation was replicated in a second independent panel of 440 M. tuberculosis strains collected during a population-based study in the Canton of Bern between 1991 and 2011. In summary, these findings support a model for TB in which the stable relationship between the human host and its locally adapted M. tuberculosis is disrupted by HIV infection.

Molecular typing of mycobacteria isolated from extrapulmonary tuberculosis patients at Debre Birhan Referral Hospital, central Ethiopia.

Abstract Background: Extrapulmonary tuberculosis (EPTB) constitutes about 10% to 20% of all cases of tuberculosis in immunocompetent patients and more than 50% of the cases in HIV-positive individuals worldwide. Little information is available on the clonal diversity of Mycobacterium species in Ethiopia from EPTB. Methods: This study was carried out on smear-negative EPTB patients to molecularly characterize Mycobacterium tuberculosis complex strains. A questionnaire, smear staining, culture, deletion typing, and spoligotyping were employed. Results: The proportional distribution of EPTB and isolates did not vary substantially (p > 0.05) amongst the socio-demographic parameters considered in the current investigation. Out of 98 fine needle aspirates processed for culture, 36.7% (36/98) were positive for mycobacterial growth. Further speciation of those culture-positive isolates showed that 88.9% were M. tuberculosis and the remaining could be non-tuberculous mycobacterial species. Spoligotyping revealed 16 clusters out of which 2 were new to the SITVIT database. The most dominant spoligotypes were SIT54, SIT53, and SIT149 in decreasing order. SIT54, SIT134, SIT173, SIT345, SIT357, SIT926, SIT91088, and SIT1580 were reported for the first time in Ethiopia. The family with the highest frequency identified was M. tuberculosis family T1, followed by family 33. Most of the strains belonged to Euro-American (61.4%) and Indo-Oceanic (36.3%) lineages. Conclusions: The present study shows the importance of M. tuberculosis as a major cause of EPTB in the study area. Moreover, the majority of isolates of M. tuberculosis were found in clusters, suggesting the possibility of the existence of recent transmission. This warrants strengthening of the control programs for EPTB in the study area.

Diagnostic challenges of single plaque-like lesion paucibacillary leprosy

The diagnosis of single-lesion paucibacillary leprosy remains a challenge. Reviews by expert dermatopathologists and quantitative polymerase chain reaction (qPCR) results obtained from 66 single-plaque biopsy samples were compared. Histological findings were graded as high (HP), medium (MP) or low (LP) probability of leprosy or other dermatopathy (OD). Mycobacterium leprae-specific genes were detected using qPCR. The biopsies of 47 out of 57 clinically diagnosed patients who received multidrug therapy were classified as HP/MP, eight of which were qPCR negative. In the LP/OD (n = 19), two out of eight untreated patients showed positive qPCR results. In the absence of typical histopathological features, qPCR may be utilised to aid in final patient diagnosis, thus reducing overtreatment and delay in diagnosis.

Clinical and epidemiological profiles of individuals with drug-resistant tuberculosis

Drug-resistant tuberculosis (TB) is a growing global threat. Approximately 450,000 people developed multidrug-resistant TB worldwide in 2012 and an estimated 170,000 people died from the disease. This paper describes the sociodemographic, clinical-epidemiological and bacteriological aspects of TB and correlates these features with the distribution of anti-TB drug resistance. Mycobacterium tuberculosis (MT) cultures and drug susceptibility testing were performed according to the BACTEC MGIT 960 method. The results demonstrated that MT strains from individuals who received treatment for TB and people who were infected with human immunodeficiency virus were more resistant to TB drugs compared to other individuals (p < 0.05). Approximately half of the individuals received supervised treatment, but most drug-resistant cases were positive for pulmonary TB and exhibited positive acid-fast bacilli smears, which are complicating factors for TB control programs. Primary healthcare is the ideal level for early disease detection, but tertiary healthcare is the most common entry point for patients into the system. These factors require special attention from healthcare managers and professionals to effectively control and monitor the spread of TB drug-resistant cases.

Diagnosis of latent tuberculosis in patients with systemic lupus erythematosus: T.SPOT.TB versus tuberculin skin test.

Early studies in patients with systemic lupus erythematosus (SLE) reported increased incidence of tuberculosis. The tuberculin skin test (TST) is the technique of choice to detect latent tuberculosis infection (LTBI) but has several limitations. OBJECTIVES We compared TST and the newer T.SPOT.TB test to diagnose LTBI in SLE patients. METHODS In this observational cohort study conducted between August 2009 and February 2012, we recruited 92 patients from those attending the SLE clinic of our university hospital. Data recorded were epidemiological and sociodemographic characteristics. Laboratory analyses included TST and T.SPOT.TB tests. RESULTS Of the patients studied, 92% were women with an average age of 42.7 years. Overall, the degree of correlation between the two tests was low (Kappa index = 0.324) but was better in patients not receiving corticosteroids (CTC)/immunosuppressive (IS) therapy (Kappa = 0.436) and in those receiving hydroxychloroquine (Kappa = 0.473). While TST results were adversely affected by those receiving CTC and/or IS drugs (P = 0.021), the T.SPOT.TB results were not. CONCLUSION Although the TST test remains a useful tool for diagnosing LTBI in SLE patients, the T.SPOT.TB test is perhaps better employed when the patient is receiving CTC and/or IS drugs.

Polymerase chain reaction detection of LeishmaniaDNA in skin biopsy samples in Sri Lanka where the causative agent of cutaneous leishmaniasis is Leishmania donovani

Leishmania donovani is the known causative agent of both cutaneous (CL) and visceral leishmaniasis in Sri Lanka. CL is considered to be under-reported partly due to relatively poor sensitivity and specificity of microscopic diagnosis. We compared robustness of three previously described polymerase chain reaction (PCR) based methods to detectLeishmania DNA in 38 punch biopsy samples from patients presented with suspected lesions in 2010. Both, Leishmaniagenus-specific JW11/JW12 KDNA and LITSR/L5.8S internal transcribed spacer (ITS)1 PCR assays detected 92% (35/38) of the samples whereas a KDNA assay specific forL. donovani (LdF/LdR) detected only 71% (27/38) of samples. All positive samples showed a L. donovani banding pattern upon HaeIII ITS1 PCR-restriction fragment length polymorphism analysis. PCR assay specificity was evaluated in samples containing Mycobacterium tuberculosis, Mycobacterium leprae, and human DNA, and there was no cross-amplification in JW11/JW12 and LITSR/L5.8S PCR assays. The LdF/LdR PCR assay did not amplify M. leprae or human DNA although 500 bp and 700 bp bands were observed in M. tuberculosis samples. In conclusion, it was successfully shown in this study that it is possible to diagnose Sri Lankan CL with high accuracy, to genus and species identification, using Leishmania DNA PCR assays.

Effects of the encapsulation of usnic acid into liposomes and interactions with antituberculous agents against multidrug-resistant tuberculosis clinical isolates

Mycobacterium tuberculosis (Mtb) has acquired resistance and consequently the antibiotic therapeutic options available against this microorganism are limited. In this scenario, the use of usnic acid (UA), a natural compound, encapsulated into liposomes is proposed as a new approach in multidrug-resistant tuberculosis (MDR-TB) therapy. Thus the aim of this study was to evaluate the effect of the encapsulation of UA into liposomes, as well as its combination with antituberculous agents such as rifampicin (RIF) and isoniazid (INH) against MDR-TB clinical isolates. The in vitro antimycobacterial activity of UA-loaded liposomes (UA-Lipo) against MDR-TB was assessed by the microdilution method. The in vitro interaction of UA with antituberculous agents was carried out using checkerboard method. Minimal inhibitory concentration values were 31.25 and 0.98 µg/mL for UA and UA-Lipo, respectively. The results exhibited a synergistic interaction between RIF and UA [fractional inhibitory concentration index (FICI) = 0.31] or UA-Lipo (FICI = 0.28). Regarding INH, the combination of UA or UA-Lipo revealed no marked effect (FICI = 1.30-2.50). The UA-Lipo may be used as a dosage form to improve the antimycobacterial activity of RIF, a first-line drug for the treatment of infections caused by Mtb.

Leprosy in children and adolescents under 15 years old in an urban centre in Brazil

This original study describes the intra-urban distribution of cases of leprosy in residents under 15 years old in Salvador, Bahia, Brazil; the study also identifies the environment in which Mycobacterium leprae is being transmitted. The cases were distributed by operational classification, clinical forms, type of contact and the addresses were geo-referenced by neighborhood. Between 2007 and 2011, were reported 145 cases of leprosy in target population living in Salvador, corresponding to detection rates of 6.21, 6.14, 5.58, 5.41 and 6.88/100,000 inhabitants, respectively. The spatial distribution of the disease was focal. Of the 157 neighborhoods of Salvador, 44 (28.6%) notified cases of leprosy and in 22 (50%) of these were detected more than 10 cases per 100,000 inhabitants. The infectious forms were found in 40% of cases. Over 90% of cases had been living in Salvador for more than five years. Overall, 52.6% reported having had contact with another infected individual inside the household and 25% in their social circle. In Salvador, M. leprae transmission is established. The situation is a major concern, since transmission is intense at an early age, indicating that this endemic disease is expanding and contacts extend beyond individual households.

UV-fixed-thick-blotch preparation improves sensitivity of auramine staining.

We describe here a very simple modification of the auramine staining procedure based on preparation of a UV-fixed thick blotch which allowed us to reach an overall sensitivity of 0.82 (592 acid-fast bacillus [AFB]-positive specimens/722 initial respiratory specimens with positive mycobacterial culture) and sensitivities of 0.93 (526 AFB-positive specimens/564 culture-positive specimens) for Mycobacterium tuberculosis complex and 0.42 (66 AFB-positive specimens/158 culture-positive specimens) for nontuberculous mycobacteria.