Over the last 30 years, there have been at least 17 published reports of nontuberculous mycobacteria (NTMs) being isolated from hospital ice or ice-making machines. Of these, 12 were reports of pseudo-outbreaks, i.e., the nosocomial transmission of organism from hospital ice/ice machines to patients, resulting in patient colonization, but with no disease manifestations. In addition, there were five outbreaks that resulted in clinical disease/pathology associated with NTM organism. Eleven different species of NTMs have been associated with these reports, where over half (59%) of the species identified were Mycobacterium fortuitum (18%), Mycobacterium gordonae (14%), Mycobacterium mucogenicum (14%), and Mycobacterium porcinum (14%). Several of these reports clearly documented that ice machines had been properly maintained, cleaned, and serviced in accordance with the CDC guidelines yet became contaminated with NTM organisms. These reports frequently detail that after extensive cleaning/disinfection following the discovery of NTM organisms, ice machines remained contaminated with NTM organisms, highlighting the difficulty in eradicating these from ice machines, once contaminated. Several reports identified that the only remedy to the contamination problem was to replace the ice machine with a new machine. Two qualitative risk assessment models are presented for (i) patients exposed to contaminated ice machine but before NTM colonization/infection and (ii) patients already colonized with NTMs from ice machines. Therefore, to protect immunocompromised/immunosuppressed patients' safety, especially during surgical or respiratory procedures, ice should not be sourced from the ice machine but should be made from sterile water and stored safely and separately away from the ice machine.

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Immunological testing for tuberculosis has been one of the most rapidly developing areas in the last decade. A new-generation immunological skin test, Diaskintest (DST), has been developed in the Russian Federation and successfully implemented into clinical practice since 2009. This article presents the results of a meta-analysis of publications reporting data on the use of the recombinant tuberculosis allergen DST (n = 121) from 2009 to 2019 included in Russian and international databases. The analysis included a total of 61 papers consistent with the study design, which cumulatively presented the results of 3,777,083 patients tested with DST (83.0%). The obtained data showed that the overall diagnostic sensitivity of the test in this population, regardless of age, was 86.0%, with 98.0% negative results. It was found that the intensity of the immune response of tuberculosis patients to specific ESAT-6 and CFP-10 antigens of DST may depend on the biological properties of the pathogen characteristic to various Mycobacterium tuberculosis genotypes, tuberculosis severity, and the presence of concomitant diseases. These factors are more prevalent in the adult population. In children, however, the test sensitivity reaches 100%. The proportion of positive DST results in HIV-positive patients tested for tuberculosis was 60.0%. The analysis showed that the accuracy (overall validity) of DST was 95.1% in the total studied population (95% confidence interval [CI]: 95.06–95.1) and 92.4% in HIV-positive patients (95% CI: 91.9–92.7).

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Leprosy is a chronic infectious disease with varied presentation. Hypopigmented or erythematous patches and plaques, skin-colored nodules, and diffuse cutaneous infiltration are the different types of cutaneous lesions seen among patients of leprosy. Verrucous lesions are an uncommon finding of the disease and may be misdiagnosed in the present times due to its rarity. We have herein described two such patients, one of whom had verrucous lesions as the only evident manifestation of leprosy, while the other developed verrucous plaque as the part of leprosy relapse.

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Background: Cutaneous tuberculosis (TB) forms a small subset of extrapulmonary TB and continues to be a significant diagnostic dilemma in routine practice. The present study is an attempt to find the incidence, clinical spectrum, and histopathological features of cutaneous TB in western Rajasthan. The relation of cutaneous TB with the human immunodeficiency virus (HIV) was also assessed. Method: A total of 40 cases of newly diagnosed patients of cutaneous TB attending the dermatology outpatient department over a period of 1 year were included in the study. A detailed clinical examination and investigations including histopathological examination were carried out. Results: The overall incidence of cutaneous TB was 0.025% (40 of 160,000 outpatients). HIV concurrence was 5% (2 cases) of all cutaneous TB cases. The most common variants were scrofuloderma (40%), lupus vulgaris (30%), TB verrucosa cutis (8%), orificial TB (2%), and lichen scrofulosorum (2%). Males suffered more than females (2.07:1) and all patients belonged to lower socioeconomic class. The Mantoux test was positive in 65% of cases. Extracutaneous involvement occurred in 17 (42.50%) cases. Characteristic well-defined tuberculoid granulomas were seen in 60% of cases, whereas 40% of cases showed nonspecific changes. Conclusion: This study provides the epidemiological data of cutaneous TB in western Rajasthan, identifies the clinicohistopathological pattern, and calls the attention of the health-care professionals that they should improve the propaedeutics of neglected and underdiagnosed cases of cutaneous TB that is prevalent in the lower socioeconomic group. Due to the varied clinical presentations, physician awareness and a high index of suspicion are necessary to diagnose cutaneous forms of TB.

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Background: Tuberculosis (TB) is still a significant health problem worldwide. Central nervous system TB amounts to 10% of all cases of TB. Despite advances in the pharmacological management of TB, the overall outcomes remain poor with significant mortality and morbidity. There are no predictors for neurological outcomes in tuberculosis meningitis (TBM). In this study, we aimed to evaluate the role of cerebrospinal fluid (CSF) C-reactive protein (CRP) in predicting mortality and neurological outcome in TBM. Method: In this observational study, all patients with TBM were recruited prospectively over a 12-month duration. Baseline demographic data, laboratory parameters, and Imaging findings were collected. CSF CRP was obtained on the CSF sample collected at the time of diagnosis. Patients were followed up at 3 months to assess neurological status and mortality. Results: Seventy-one patients with TBM were recruited in this study. The overall mortality in this study was 22.5% of patients. The primary composite outcome of mortality and adverse neurological outcome occurred in 40.8%. The CSF CRP levels ranged between 0.1 and 4.8 mg/dl, and the mean CSF CRP level was 1.11 mg/dl. The Relative risk for a patient with high CSF CRP to develop adverse outcome was 1.84 (P = 0.038). CSF CRP was a good predictor of mortality with a relative risk of 2.92 (P = 0.027). Stroke in TBM had a high incidence of 47.9% and a relative risk of 3.42 for an adverse neurological outcome. CSF CRP did not predict the occurrence of stroke. Hydrocephalus and elevated intracranial pressure were good predictors of stroke. Conclusion: TBM is a disease with significant mortality and morbidity. CRP level in the CSF can be measured, but a highly sensitive scale may be needed as the mean values were much lower compared to the serum values. CSF CRP Levels showed significant associations with adverse outcomes and mortality.

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Background: There are few studies investigating the prevalence of latent tuberculosis infection (LTBI) in HIV-1-infected children on antiretroviral therapy (ART), but no data from Nigeria. This study determined the prevalence of LTBI in HIV-1-infected children on ART in our clinic. Knowing the prevalence and thus the burden of LTBI could help improve HIV care by enabling targeted isoniazid (INH) prophylaxis. Method: This observational study was carried out from September 2016 to August 2017 at the pediatric HIV clinic of the Jos University Teaching Hospital among HIV-1-infected children on ART, aged 6 months–15 years. LTBI was diagnosed using an interferon-gamma release assay, the ELISpot test, T-SPOT®.TB assay (Oxford Immunotec, Abingdon, UK) on freshly collected whole blood samples within 2 h. Children with a positive test were treated with INH after first excluding TB by chest X-ray and clinical evaluation. Results: Of the 90 children studied, 4 (4.4%) had LTBI diagnosed by ELISpot. Their median interquartile range (IQR) age was 10.4 years (7.9–12.5), the majority were male (54.4%) and most of them had originally received Bacille Calmette-Guérin (83/89, 93.3%). They had a median CD4 count of 694 cells/μL (472–1045). The median (IQR) CD4 count was higher in LTBI compared to non-LTBI children: 1286 cells/μL (953–1375) versus 683 cells/μL (465–1040), (P = 0.044). Conclusion: Although this study showed a very low prevalence of LTBI in our setting, it was still beneficial to the few children on ART identified with LTBI as it enabled treatment with INH. A larger study will be required to ascertain the actual burden of LTBI in such children in our setting.

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Background: The objective of this study is to determine the initial drug resistance pattern among new tuberculosis (TB) cases and assess the extent of association with human immunodeficiency virus (HIV) and diabetes mellitus (DM). Method: This is a retrospective analysis of 1116 clinical isolates were collected from patients who were newly diagnosed with TB at TB Laboratory between January 2016 and November 2019 and used for determining drug-resistance profiles against five first-line and five second-line anti-TB drugs; and the results were assessed the association between TB risk factors and primary drug resistance TB. Results: Of the 1116 newly diagnosed TB patients, 193 (17.3%) showed resistance to at least one or more of the first-line drugs by different patterns, 105 (9.4%) showed resistance to one drug, 38 (3.40%) showed polyresistance, 50 (4.5%) showed multidrug resistant (MDR), and one patient had extensively drug resistant. Mono-resistance to isoniazid (INH), STR, pyrazinamide, and rifampicin were seen in 40 (3.6%), 33 (2.95%), 29 (2.59%), and 3 (0.3%) of isolates, respectively. INH showed the highest percentage of resistance among the patients. Of 1116 newly diagnosed TB patients, 256 (22.9%) were TB-DM cases and 135 (12.9%) were TB-no DM cases. The rates of drug resistance-TB 46/1116 (4.12%), monoresistance 25 (2.24%), polyresistance 9 (0.8%), and MDR 12 (1.07%) among TB-DM group were higher than TB-no DM group. Conclusion: Our study confirms that resistance to INH was the most common phenomenon. We found that diabetes was identified as a risk factor of TB drug resistance. We did not find a significant association between HIV co-infection and TB drug-resistance

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Background: Heteroresistance is the coexistence of susceptible and resistant strains in the same individual, considered the preliminary step for total resistance, and can stem from mixed infection or clonal heterogeneity. The aim of this study was to evaluate the heteroresistance of Mycobacterium tuberculosis to rifampicin and isoniazid and its characterization. Method: GenoType MTBDRplus^®; Sanger sequencing of the rpoB, katG, and inhA genes; and Mycobacterial Interspersed Repetitive Unit – Variable Number Tandem Repeat (MIRU-VNTR) were performed. Results: In a total of 654 isolates, 530 were resistant, 124 were susceptible, and 29 were heteroresistant to a first-line drug. GenoType MTBDRplus^® detected heteroresistance in the rpoB gene in 26/29 (89.6%), as compared to 5/29 (17.2%) in the katG gene and 2/29 (6.8%) in the inhA gene. Four isolates showed heteroresistance in these genes. The Sanger sequencing detected heteroresistance in the rpoB gene in 7/29 (24.1%), as compared to 3/29 (10.3%) in the katG gene. In one isolate, heteroresistance was concomitant in both the rpoB and katG genes. MIRU-VNTR detected mixed infection in three heteroresistant isolates, while four isolates showed clonal heterogeneity. Conclusions: GenoType MTBDRplus^® detected more cases of heteroresistance when compared to sequencing. It was also possible to characterize mixed infection and clonal heterogeneity by MIRU-VNTR.

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Cutaneous nodules in children arise from variety of pathologic conditions. Thorough history taking and clinical examination helps in narrowing down the differential diagnosis and reducing the need for an extensive panel of investigatons. Here, we report a case of cutaneous nodules in an adolescent male which posed diagnosed challenge and finally diagnosed with cytology.

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Background: The diagnosis of tuberculosis (TB) has mostly been relied on a long-used method called sputum smear microscopy. In 2010, Xpert MTB/RIF assay was approved by the World Health Organization for simultaneous TB diagnosis and detection of resistance. Our current study was undertaken to compare the diagnostic performance of Xpert MTB/RIF assay to auramine staining-based light-emitting diode-Fluorescence Microscopy (LED-FM) considering culture as the gold standard method for pulmonary and extrapulmonary TB. Method: Pulmonary and extrapulmonary specimens of suspected TB patients were examined in this study. From January 2016 to June 2019, sputum, urine, superficial swabs, gastric aspirates, and pleural infusion specimens were collected from new and previously treated TB individuals. Specimens were examined using Xpert MTB/RIF, LED-FM, and Mycobacterium culture techniques to evaluate their performance. Results: A total of 697 suspected TB samples were included in this analysis, and of these, 469 (67.29%) were positive for all three used methods. The overall sensitivities, specificities, and positive and negative predictive values were 99.6%, 62.0%, 88.4%, and 98.2% for Xpert MTB/RIF and 88.0%, 95.6%, 99.0%, and 60.7% for LED-FM, respectively, compared to culture method. Conclusion: The sensitivity of Xpert MTB/RIF assay was observed to be higher than the LED-FM method, thus suggesting this molecular technique as a promising tool for the diagnosis of pulmonary and extrapulmonary TB, which will help in the management of TB infections in developing countries such as Mali.

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Background: The disease severity in pulmonary Multidrug-resistant tuberculosis (MDR-TB) varies from mild to severe, which is determined by host and pathogen virulence factors. The difference of symptoms felt by TB patients were interesting to investigate in discovering whether its the human immune response or bacteria's virulence gene that plays the role. The aim of this research was to analyze association between disease severity degree of pulmonary MDR-TB patients with Single nucleotide polymorphisms (SNPs) found in toll-like receptors (TLRs) gene. Method: Blood samples were obtained from pulmonary MDR-TB patients in Dr. Soetomo Hospital, Surabaya, Indonesia. Polymerase chain reaction (PCR) multiplex and target SNPs were analyze using DigiTag2 assay. The variant of esxA gene was determined using PCR and sequencing. Severity degree was determined by chest X-ray, the lesions were scored according to their severity, score of =2.5 ranking as mild, 2.5–6 as moderate and =6 as severe. Association level between SNP in TLRs gene degree of pulmonary MDR-TB was analyzed using Chi-square test. Bonferroni correction for multiple comparison was used to anticipate genotyping error. Results: A total of 22 MDR-TB patients were classified into severe degree group, while 16 patients were moderate/mild degree. SNPs in encoding gene of TLRs were mostly found in intron, specifically in TLR-1, TLR-2, and TLR-6. HWE P value in rs5743572 was 0.841; in rs3804100 was 0.0176; and in rs5743808 was 0.562. Association analysis between SNP in TLRs genes and degree of disease revealed significant association in rs5743572, SNP of TLR-1, with P < 0.05; odds ratio [OR] = 11.67 (95% confidence interval [CI]: 3.94–34.52); rs3804100, SNP of TLR-2 had P < 0.05; OR = 37.59 (95% CI: 9.30–151.88); and rs5743808, the SNP of TLR-6 had P < 0.05; OR = 31.5 (95% CI: 8.60–115.34). Conclusions: We concluded that SNPs in TLR-1, TLR-2, and TLR-6 of pulmonary MDR-TB patients was found to have an association with disease severity. TLRs polymorphism had significant association was present in TLR-1 rs5743572 in intron, TLR-2 rs3804100 in exon, and TLR-6 rs5743808 in exon and among MDR-TB isolates from patients with pulmonary MDR-TB of severe and moderate/mild degree.

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In pulmonary practice, pleural effusion is a commonly encountered entity and has various etiologies. Pleural effusions in postpartum women can be an incidental self-limiting finding. The presence of systemic or respiratory symptoms, however, calls for prompt etiological workup and targeted therapy. Tuberculous pleuritis and lupus-related pleural disease are well known to flare up in the postpartum period. We describe the case of a young healthcare worker with no previous comorbidities who presented with fever, breathlessness, and chest pain 2 weeks after an uneventful confinement. Chest radiograph revealed moderate left-sided pleural effusion. Pleural fluid analysis was biochemically consistent with tubercular effusion. Pleural biopsy histological examination showed features of xanthomatous pleuritis and Cartridge based nucleic acid amplification test (CB-NAAT) showed evidence of Mycobacterium tuberculosis(MTB). She was initiated on antitubercular medicines to which she responded well with the resolution of clinical symptoms and pleural collection. This is the first case report describing an association of xanthogranulomatous pleuritis with tuberculosis.

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Reactive perforating leprosy (RPL) is a rare clinical manifestation of type 2 leprosy reaction. A case of lepromatous leprosy (LL) with type 2 leprosy reaction presented as RPL in one patient was reported. A physical examination showed multiple punched-out ulcers with regular border, without undermined edge, and dermal base. The ulcers mostly covered with hemorrhagic crust, contained pus, and necrotic tissue. A histopathological examination revealed invagination of the epidermis, intracorneal abscess, and infiltration of foamy macrophages with lymphocytes in dermis that supported the diagnosis of LL with RPL. The patient was given multidrug therapy-multibacillary and 40 mg prednisone daily which tapered off and wound dressing. Clinical improvement was observed within 2 weeks of treatment, as some ulcers healed. Type 2 leprosy reaction can provide a variety of clinical manifestations, one of which is RPL.

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Gastrointestinal tuberculosis usually involves ileum and cecum in three-fourth of cases. Isolated gastric involvement is uncommon in the absence of pulmonary tuberculosis or immunodeficiency in affected individuals. Here, we describe a case of tuberculosis involving stomach and colon in an immunocompetant young patient who presented to us with dyspeptic symptoms, pain abdomen, and melena. Morphologically, the lesion mimicked as advanced malignancy, but laparoscopic biopsy confirmed the diagnosis. The patient responded well to medical treatment. It should be emphasized that tuberculosis can involve any part of gastrointestinal tract including stomach even in immunocompetent individuals, and it should be kept as differential diagnosis of any chronic inflammatory lesion of stomach, especially in endemic countries as medical treatment is usually sufficient to provide a cure.

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A 74-year-old female was hospitalized for further study of chest computed tomography (CT) scan compatible with mycobacterial infection. She had no history of underlying lung disease or immune alteration. At the moment of admission, she was completely asymptomatic. Scedosporium apiospermum a filamentous fungus was isolated first in the bronchoalveolar lavage. Weeks later, Mycobacterium avium complex grows in the mycobacterial culture sample.

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Background: Levofloxacin is a preferred drug for multidrug-resistant (MDR)-tuberculosis (TB) with bactericidal activity that correlates with the pharmacokinetic exposures of serum peak concentration (C_max) and total area under the concentration time curve (AUC_0-24). Pharmacokinetic exposures can be measured to personalize dosing to reach targets, but this practice requires venepuncture, chromatographic or mass spectrometry equipment, and technical expertise. We sought to demonstrate the accuracy of using urine colorimetry as a more feasible estimation of levofloxacin exposure. Method: A colorimetric method using bromocresol green was tested on spiked urine samples with levofloxacin measured using a spectrophotometer. This method was tested in urine samples of healthy volunteers given one 750 mg dose of levofloxacin with urine collected at 0–4 h, 4–8 h, and 8–24 h intervals, and concomitant serum samples were collected and analyzed by high-performance liquid chromatography. Validation of this assay was done in a cohort of people living with human immunodeficiency virus (PLWH), initiating a levofloxacin containing MDR-TB regimen. Results: Urine colorimetry was reproducible in spiked samples and the calibration was curve linear for levofloxacin concentrations ranging from 7.8 μg/ml to 250 μg/ml, with r = 0.98. In healthy volunteers, correlation between urine absorbance values and serum AUC_0-24 was highest in urine collected between 4 and 8 h (r = 0.91, P = 0.01), yet in PLWH, urine collected between 0 and 4 h had highest correlation (r = 0.66, P = 0.05). The area under the receiver operating characteristics curve was >0.8 in the derivation, as well as the validation cohort for the urine absorbance values identifying people with total serum exposure below target. Conclusion: Urine colorimetry was highly sensitive in predicting target serum concentrations. Colorimetric methods to determine levofloxacin in urine may improve the feasibility of therapeutic drug monitoring and personalized dose adjustment in TB endemic settings.

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Background: Mycobacterium tuberculosis is able to survive and persist as an intracellular pathogen by modulating its own metabolism and host immunity. The molecules and mechanisms utilized to accomplish this modulation are not fully understood. The present study elucidates the effects of M. tuberculosis secretory antigens on T-cell-receptor (TCR)/CD28-triggered signaling in Jurkat T-cells. Method: In the present study, intracellular calcium mobilization was investigated in CD3-activated cells in response to M. tuberculosis antigens, Ag85A, early secretory antigenic target-6 (ESAT-6), and H₃₇Rv. The activation of mitogen-activated protein kinases, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and p-38 was also analyzed in CD3- and CD28-activated cells by western blotting. Results: Our results showed CD3-triggered modulations in free intracellular calcium levels in Jurkat T-cells in response to M. tuberculosis antigens. In addition, we also noted M. tuberculosis antigens induced downregulation in phosphorylation of ERK1/2 and p-38. Overall, our results proposed that M. tuberculosis secretory antigens, particularly ESAT-6, impede TCR/CD28-induced signaling events which could be responsible for T-cell unresponsiveness, implicated in the progression of disease. Conclusion: The present study demonstrated M. tuberculosis secretory antigens induced alteration of T-cell signaling pathways in unsensitized Jurkat T-cell line which might be implied in T-cell dysfunctioning during the progression of the disease.

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Tuberculosis (TB) is known for its varied presentation and complications, the most dreaded complication being central nervous system (CNS) TB which includes tuberculoma. We present a case report of an asymptomatic recurrent case of CNS tuberculoma requiring multiple surgeries and prolonged critical care management.

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Tuberculosis (TB) is the leading infectious cause of death worldwide. Tubercular meningitis is the most severe form of extrapulmonary TB which carries high morbidity and mortality. Intracranial tuberculoma may develop paradoxically during the treatment of tubercular meningitis, which is a quite rare occurrence. There are many reported cases of paradoxical development of optochiasmatic tuberculoma during the treatment of tubercular meningitis, causing severe visual impairment. Association of Guillain-Barré syndrome with TB is a rare entity. However, the co-occurrence of optochiasmatic tuberculoma and Guillain-Barré syndrome is even rarer and not reported before in literature. Here, we present the first case of co-occurrence of optochiasmatic tuberculoma and Guillain-Barré Syndrome, developing as a paradoxical reaction in a 23-year-old male.

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Background: Tuberculosis and malaria (TB/MP) co-infection generates severe pathology that affects the levels of cytokines and hemostatic parameters than either disease. Anti-TB treatment regimen involves phases of different drug cocktails that may additionally modulate the levels of inflammatory cytokines and hemostatic parameters. This study investigated the variations in the levels of hemostatic and inflammatory markers when compared between TB patients with and without malaria at pretreatment, intensive, and continuation phase treatment. Method: In this cross-sectional study, 180 patients were recruited comprising; 35 TB-only and 25 TB/malaria patients at pretreatment, 36 TB-only and 24 TB/malaria patients at intensive phase treatment, and 27 TB-only and 33 TB/malaria patients at continuation phase therapy. P-selectin (P-SEL), platelet-activating factor (PAF), platelet factor-4, GP IIb/IIIa complex, tumor necrosis factor-alpha (TNF-α), interleukin (IL)-10, IL-6, IL-2, transforming growth factor (TGF)-β, and thrombopoietin (TPO) were assayed using enzyme-linked immunosorbent assays. Mann–Whitney test and Spearman's rank correlation were applied for statistical test. Results: At pretreatment, the median levels of IL-6 and IL-10 were significantly lowered, while P-selectin (P-SEL), GP IIb/IIIa, and PAF were significantly increased in TB/malaria patients compared to TB patients without malaria. At intensive treatment, TNF-α, IL-6, and IL-2 were significantly higher, while IL-10 and PAF were significantly reduced in TB/malaria patients compared with TB patients without malaria. At continuation phase treatment, TNF-α, IL-6, TGF-β, PF4, GP IIb/IIIa, and TPO were significantly reduced, while P-SEL was significantly increased in TB/malaria patients compared with TB patients without malaria. Conclusion: Differences in the levels of inflammatory cytokines and hemostatic markers between TB patients co-infected with malaria and nonmalaria-infected TB patients vary with anti-TB treatment.

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Invasive pulmonary aspergillosis (IPA) is a life-threatening condition. Patients with lung cancer 73.9% died within 1 month after IPA diagnosis. Diagnosing IPA is challenging because of the nonspecific clinical symptoms, radiological findings, lack of sensitivity, and need time in culture method. A 73-year-old male presented with shortness of breath, productive cough, fever, chest pain, and a decrease of body weight. On right thorax auscultation, decreased of vesicular breath sound, rhonchi, and pleural friction rub were found. The chest radiograph revealed a right lung tumor. We confirmed the existence of Aspergillosis on the fiberoptic bronchoscopy (FOB) result by conducting the serology examination (1,3-β-D-glucan and Galactomannan) using the enzyme-linked immunosorbent assay method. Bronchoalveolar lavage culture from FOB result was Aspergillus fumigatus, and fine-needle aspiration biopsy showed adenocarcinoma. Because IPA is a life-threatening condition, an early diagnosis is important. Therefore, a serology test is necessary for the early detection of suspected case of fungal infection and useful to complement the culture.

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Background: Bovine tuberculosis (bTB) is a zoonotic disease caused by Mycobacterium bovis that mainly affects cattle. Although vaccination is the most effective strategy to control bTB, it may interfere with the diagnosis of the infection. Therefore, ancillary tests to differentiate vaccinated from infected animals (DIVA) are essential in a cattle vaccination scenario. ESAT-6 and CFP-10 are the most promissory DIVA antigens. Method: In this study, we deleted esat6 and cfp10 genes from the M. bovis Δ mce2 live-attenuated vaccine candidate and evaluated its protection level against bTB in BALBc mice. Results: We found that the M. bovis strain mutant in mce2, esat-6 and cfp-10 failed to confer protection against virulent M. bovis challenge in a mouse model of tuberculosis. Conclusions: This result highlights the relevant role of ESAT-6 and CFP-10 in the induction of protective immune response against M. bovis infection and reveals the need of evaluating different strategies to compensate for the lack of these DIVA antigens in new vaccine formulations.

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