Follow-up assessment of patients with Pure Neural Leprosy in a reference center in Rio de Janeiro—Brazil

IntroductionPure Neural Leprosy (PNL) is a rare clinical form of leprosy in which patients do not present with the classical skin lesions but have a high burden of the disability associated with the disease. Clinical characteristics and follow up of patients in PNL are still poorly described in the literature.ObjectiveThis paper aims to describe the clinical, electrophysiological and histopathological characteristics of PNL patients, as well as their evolution after multidrug therapy (MDT).MethodsFifty-two PNL patients were selected. Clinical, nerve conduction studies (NCS), histopathological and anti-PGL-1serology were evaluated. Patients were also assessed monthly during the MDT. At the end of the MDT, all of the patients had a new neurological examination and 44 were submitted to another NCS.ResultsParesthesia was the complaint most frequently reported by patients, and in the neurological examination the most common pattern observed was impairment in sensory and motor examination and a mononeuropathy multiplex. Painful nerve enlargement, a classical symptom of leprosy neuropathy, was observed in a minority of patients and in the motor NCS axonal injuries, alone or in combination with demyelinating features, were the most commonly observed. 88% of the patients did not present any leprosy reaction during MDT. There was no statistically significant difference between the neurological examinations, nor the NCS pattern, performed before and after the MDT.DiscussionThe classical hallmarks of leprosy neuropathy are not always present in PNL making the diagnosis even more challenging. Nerve biopsy is an important tool for PNL diagnosis as it may guide therapeutic decisions. This paper highlights unique characteristics of PNL in the spectrum of leprosy in an attempt to facilitate the diagnosis and management of these patients.     

Caffeic acid phenethyl ester modulates aflatoxin B1‐induced hepatotoxicity in rats

Aflatoxin B1 (AFB1) is the most potent of the mycotoxins and is widely observed in nutrition abnormalities. There are some studies suggesting oxidative stress‐induced toxic changes on liver related to AFB1 toxicity. The aim of the present study was to evaluate whether antioxidant caffeic acid phenethyl ester (CAPE) relieves oxidative stress in AFB1‐induced liver injury in rat. Twenty‐four male rats were equally divided into three groups. The first group was used as a control. The second group received three doses of AFB1. The three doses of CAPE were given to constitute the third group with doses of AFB1. After 10 days of experiment, liver and serum samples were taken from all animals. Serum gamma glutamyl transferase (GGT), alkaline phosphatase (ALP), glutathione s‐transferase (GST), nitric oxide (NO) and sulfhydryl values were higher in the AFB1 group than in control, whereas serum GGT, ALP, GST and NO values were decreased by in the AFB1 + CAPE group than in AFB1 group. Liver GST, total oxidant capacity, sulfhydryl, apoptosis index and ischemia‐modified albumin values were higher in the AFB1 group than in control, whereas the GST activity and apoptosis index were lower in the AFB1 + CAPE group than in the AFB1 group. There were histopathological degeneration and apoptosis in hepatocytes of AFB1 group. The findings were totally recovered by CAPE administration. In conclusion, we observed that AFB1 caused oxidative and nitrosative hepatoxicity to hepatocytes in the rat. However, CAPE induced protective effects on the AFB1‐induced hepatoxicity by modulating free radical production, biochemical values and histopathological alterations. Copyright © 2013 John Wiley & Sons, Ltd.     

Fine needle aspiration cytology and immunocytochemistry in tuberculous thyroiditis: a case report

BACKGROUND: Tuberculosis of the thyroid is very rare and does not strike the clinician as a first clinical diagnosis of a thyroid nodule. To our knowledge, only 40 cases of tuberculous thyroiditis diagnosed by fine needle aspiration cytology (FNAC) are described in the English literature. CASE: We report a case of tuberculous thyroiditis in a young woman who presented with a right-side solitary thyroid nodule of short duration (15 days), diagnosed by FNAC and confirmed by positive immunocytochemistry with monoclonal antibody to Mycobacterium tuberculosis complex. Ziehl Neelsen staining for acid-fast bacilli (AFB) was negative. CONCLUSION: FNAC provides a confident preoperative diagnosis of thyroid tuberculosis, obviating the need for unnecessary surgical removal of thyroid nodule. Immunocytochemistry is an important diagnostic adjunct to FNAC in AFB-negative cases.     

Biomarkers for screening of lung cancer and pre-neoplastic lesions in a high risk Chilean population

Background

The mortality of lung cancer (LC), increases each year in the world, in spite of any advances, in development of new drugs to advance stages of LC. The high incidence of LC has been associated with smoking habit, genetic diversity and environmental pollution. Antofagasta region has been reported to have the highest LC mortality rate in Chile and its inhabitants were exposed to arsenic in their drinking water in concentrations as high as 870 μg/L. Non-invasive techniques such as biomarkers (Automatic Quantitative Cytometry: AQC and DR70) and Auto Fluorescence Bronchoscopy (AFB) might be potentially useful as a supplementary diagnostic approach and early detection. Early detection is one of the most important factors to intervene and prevent cancer progression in LC. This is a work of an ongoing prospective bimodality cancer surveillance study in high risk LC volunteers. Enrolment was done in subjects from Antofagasta and Metropolitan regions. In addition, we enrolled subjects who were suspected of having lung cancer. AQC, DR70 and AFB were used as tools in the detection of pre-neoplastic (PNL) and neoplastic lesions (NL).

Results

Half of the samples, classified as suspicious by AFB, were confirmed as metaplasia or dysplasia by histopathology. For LC, DR70 showed a higher sensitivity (95.8%) and specificity (91.9%) than AQC. However, for PNL AQC showed a higher sensitivity (91.9%) than DR70 (27.3%), although both with low PPV values. As a pre screener, both biomarkers might be employed as complementary tools to detect LC, especially as serially combined tests, with a sensitivity of 60% and a PPV of 65.2%. Additionally, the use of parallel combined tests might support the detection of PNL (sensitivity 91.2%; PPV 49.1%).

Conclusion

This work adds information on cellular and molecular biomarkers to complement imaging techniques for early detection of LC in Latin America that might contribute to formulate policies concerning screening of LC. Supported by INNOVA-CORFO, Chile.     

Nucleic acid amplification of Mycobacterium tuberculosis complex DNA from archival fine needle aspiration smear scrapings vs. fresh fine needle aspirates of tuberculous lymphadenitis

OBJECTIVE: To assess the efficacy of the nucleic acid amplification (NAA) technique for Mycobacterium tuberculosis (MTB) complex from archival fine needle aspirate (FNA) smear scrapings of confirmed cases of extrapulmonary tuberculosis (EPTB) for a retrospective diagnosis of EPTB as compared to NAA from fresh FNA material from the same cases. STUDY DESIGN: Smear scrapings from 51 cases; 33 cases of tuberculous lymphadenitis (from patients who had undergone NAA 1 year before for MTB from fresh FNA material); 13 negative controls from nontuberculous, archival FNA smears; and 5 known acid-fast bacilli (AFB)-positive sputum smears, were subjected to NAA using the IS6110 primer sequence of M tuberculosis. Ziehl-Neelsen staining was done in all the smears. RESULTS: Of the 33 cases of tuberculous lymphadenitis, 15 (45.4%) were AFB positive and 18 (64.5%) AFB negative. MTB NAA was positive in 73.3% (11 of 15 AFB-positive cases) in the freshly aspirated material and was observed in 60% (9 of 15 AFB-positive cases) when done on DNA extracted from the archival smear scrapings of the same cases. Similarly, in the 18 AFB-negative cases, MTB NAA positivity was 72.2% (13 of 18) on fresh material and 44.4% (8 of 18) on archival smear scrapings from the same AFB-negative cases. Overall NAA positivity was 51.5% for archival smear scrapings as compared to 71% for fresh FNA of the same cases. CONCLUSION: Low NAA sensitivity of MTB DNA in archival material of known tuberculous cases limits the routine use of NAA based retrospective molecular diagnosis of MTB complex.     

Use of fine needle aspiration cytology in leprotic lesions: a report of 4 cases

BACKGROUND: Although a few studies have shown fine needle aspiration cytology (FNAC) to be a sensitive diagnostic tool in the detection of nerve involvement, its role as an initial diagnostic procedure in pure neuritic leprosy (PNL) and in the detection of skeletal lesions with unusual findings has not been documented before. CASES: Three patients who presented with thickened nerves and a fourth with biopsy-proven lepromatous leprosy with lesions in hand bones underwent FNAC. Of the 3 patients with nerve thickening, 2 had a clinical suspicion or diagnosis of neuritic leprosy, whereas in the third patient a clinical differential diagnosis of a soft tissue tumor or parasitic cyst was considered. FNAC in all 3 cases revealed epithelioid cell granulomas, Langhans giant cells and caseous necrosis. Fites and Ziehl-Neelsen stains were negative for acid-fast bacilli. Cytologic diagnosis of pure neuritic leprosy was made in all 3 cases and confirmed by histopathologic examination. FNAC of skeletal lesions from the fourth patient confirmed involvement of bone with unusual cytologic findings of epithelioid cell granulomas and giant cells along with a significant proportion of foamy macrophages and strong Fites stain positivity. CONCLUSION: FNAC is a simple, useful, minimally traumatic and routinely applicable procedure in the diagnosis of pure neuritic leprosy and leprous osteitis.     

Distribution of Paenibacillus larvae spores inside honey bee colonies and its relevance for diagnosis

One of the most important factors affecting the development of honey bee colonies is infectious diseases such as American foulbrood (AFB) caused by the spore forming Gram-positive bacterium Paenibacillus larvae. Colony inspections for AFB clinical symptoms are time consuming. Moreover, diseased cells in the early stages of the infection may easily be overlooked. In this study, we investigated whether it is possible to determine the sanitary status of a colony based on analyses of different materials collected from the hive. We analysed 237 bee samples and 67 honey samples originating from 71 colonies situated in 13 apiaries with clinical AFB occurrences. We tested whether a difference in spore load among bees inside the whole hive exists and which sample material related to its location inside the hive was the most appropriate for an early AFB diagnosis based on the culture method. Results indicated that diagnostics based on analysis of honey samples and bees collected at the hive entrance are of limited value as only 86% and 83%, respectively, of samples from AFB-symptomatic colonies were positive. Analysis of bee samples collected from the brood nest, honey chamber, and edge frame allowed the detection of all colonies showing AFB clinical symptoms. Microbiological analysis showed that more than one quarter of samples collected from colonies without AFB clinical symptoms were positive for P. larvae. Based on these results, we recommend investigating colonies by testing bee samples from the brood nest, edge frame or honey chamber for P. larvae spores.     

PCR-Based Techniques for Leprosy Diagnosis: From the Laboratory to the Clinic

In leprosy, classic diagnostic tools based on bacillary counts and histopathology have been facing hurdles, especially in distinguishing latent infection from active disease and diagnosing paucibacillary clinical forms. Serological tests and IFN-gamma releasing assays (IGRA) that employ humoral and cellular immune parameters, respectively, are also being used, but recent results indicate that quantitative PCR (qPCR) is a key technique due to its higher sensitivity and specificity. In fact, advances concerning the structure and function of the Mycobacterium leprae genome led to the development of specific PCR-based gene amplification assays for leprosy diagnosis and monitoring of household contacts. Also, based on the validation of point-of-care technologies for M. tuberculosis DNA detection, it is clear that the same advantages of rapid DNA detection could be observed in respect to leprosy. So far, PCR has proven useful in the determination of transmission routes, M. leprae viability, and drug resistance in leprosy. However, PCR has been ascertained to be especially valuable in diagnosing difficult cases like pure neural leprosy (PNL), paucibacillary (PB), and patients with atypical clinical presentation and histopathological features compatible with leprosy. Also, the detection of M. leprae DNA in different samples of the household contacts of leprosy patients is very promising. Although a positive PCR result is not sufficient to establish a causal relationship with disease outcome, quantitation provided by qPCR is clearly capable of indicating increased risk of developing the disease and could alert clinicians to follow these contacts more closely or even define rules for chemoprophylaxis.     

Development of a new antigen detection dot-ELISA for diagnosis of tubercular lymphadenitis in fine needle aspirates

A sandwich dot enzyme-linked immunosorbent assay (ELISA) was standardized to detect mycobacterial antigen in fine needle aspirates of patients with tubercular lymphadenitis (TBLN). The assay was performed on nitrocellulose paper by using antibodies raised in mice and rabbits against crude soluble protein (CSP) of Mycobacterium tuberculosis. The test was able to detect as low as 5 ng protein/ml. A total of 225 suspected cases of tubercular lymphadenopathy were screened, out of which 96 were cytomorphologically confirmed as cases of tubercular lymphadenitis (50 acid-fast bacilli (AFB)-positive and 46 AFB-negative). These were considered as positive controls. Only 28 cases were proven to be of nontubercular etiology and were considered as negative controls. In the remaining 101 (39 scanty) aspirates, tubercular etiology could neither be ruled out nor confirmed. Out of 50 AFB-positive confirmed cases of tubercular lymphadenitis, 46 were ELISA-positive. Out of 46 AFB-negative but cytomorphologically confirmed aspirates, antigen could be demonstrated in only 42 aspirates. Four samples from patients with nontubercular etiology were also found to be ELISA-positive. Antigen was picked up in a total of 90.3% of aspirates with suspicion of tuberculosis and 79.5% of scanty aspirates. The assay was found to be 91.6% sensitive and 85.7% specific. The assay was found to be simple and rapid, and hence, could be performed in areas where health facilities are rudimentary.     

A difficult diagnosis: a case report of combined Riedel's disease and fibrosing Hashimoto's thyroiditis

Riedel's disease (RD) is a rare form of chronic thyroiditis, predominantly characterised by fibrosis which may involve neighbouring tissues and organs. Hashimoto's disease (HD), on the other hand, is fairly common. Clinical differentiation between these diseases is often difficult, and the results of diagnostic imaging, laboratory tests and cytology studies are often similar. We report the case of a female patient with Riedel's thyroiditis displaying clinical, laboratory and radiological traits of both diseases. A 44 year-old Caucasian female was diagnosed with hypothyroidism. A fine-needle aspiration biopsy was performed; the findings were suggestive of an exacerbated chronic inflammatory process. However, a small lymphocyte-derived malignancy could not be ruled out with certainty, and so the patient was referred for elective thyroidectomy. The microscopic features of both specimens did not meet the criteria of Hashimoto's thyroiditis. The immunohistochemical studies revealed few scattered B lymphocytes (CD20 positive) and numerous scattered T lymphocytes (CD3 positive). Finally, Riedel's thyroiditis with an intense inflammatory infiltrate composed of lymphocytes was diagnosed. Reaching a diagnosis was particularly difficult in this patient, since Riedel's thyroiditis, the fibrosing form of Hashimoto's disease and malignant tumours of the thyroid can show similar traits upon physical and histopathological examination. As the clinical data was indicative of Hashimoto's thyroiditis and there were partial histological criteria of two forms of thyroiditis, namely Hashimoto's and Riedel's, the very rare diagnosis of a combined disease was made. Dense B and T lymphocytes and some plasma cell infiltrates, as well as the destruction of thyroid follicles by fibrosis extending into surrounding tissues, were supportive of the eventual diagnosis. Differentiating between the histopathological and clinical presentation of both diseases in one patient is difficult, primarily due to the partial overlapping of their histopathological traits. In order to avoid a diagnostic error, close cooperation between the endocrinologist and pathologist is mandatory. It is our opinion that in our patient the two diseases existed separately, and their coexistence was most likely coincidental.     

Tuberculous orchiepididymitis, meningoencephalitis and hydrocephalus

Tuberculous meningoencephalitis (TBM) is a rare and serious, often fatal presentation of active tuberculosis and account for about 1% of cases. Early diagnosis and prompt treatment of TBM is essential to reduce morbidity and mortality. Here, we report a case of TBM in 60-year-old man. TBM was considered on the basis of clinical presentation, laboratory findings (hyponatraemia), cerebrospinal fluid studies, radiological findings (hydrocephalus on multi-slice computed tomography), and history of orchiepididymitis of unknown origin one year earlier, together with information that the patient originated from Kosovo where incidence of tuberculosis is still high. Mycobacterium tuberculosis was cultured from cerebrospinal fluid on Lowenstein-Jensen medium confirming diagnosis of TBM. Subsequently, acid-fast bacilli (AFB) staining on samples obtained after orchiectomy a year ago was performed, revealing AFB. Anti-tuberculosis therapy is still in course. This is the second case of tuberculous meningoencephalitis with the same disease pattern (i.e. tuberculous orchiepididymitis - meningoencephalitis) in our Department, and this fact was crucial for the presumptive diagnosis and urgent treatment of TBM. The former case was described five years ago.     

Association of overactive bladder and stress urinary incontinence in rats with pudendal nerve ligation injury

Approximately one-third of patients with stress urinary incontinence (SUI) also suffer from urgency incontinence, which is one of the major symptoms of overactive bladder (OAB) syndrome. Pudendal nerve injury has been recognized as a possible cause for both SUI and OAB. Therefore, we investigated the effects of pudendal nerve ligation (PNL) on bladder function and urinary continence in female Sprague-Dawley rats. Conscious cystometry with or without capsaicin pretreatment (125 mg/kg sc), leak point pressures (LPPs), contractile responses of bladder muscle strips to carbachol or phenylephrine, and levels of nerve growth factor (NGF) protein and mRNA in the bladder were compared in sham and PNL rats 4 wk after the injury. Urinary frequency detected by a reduction in intercontraction intervals and voided volume was observed in PNL rats compared with sham rats, but it was not seen in PNL rats with capsaicin pretreatment that desensitizes C-fiber-afferent pathways. LPPs in PNL rats were significantly decreased compared with sham rats. The contractile responses of detrusor muscle strips to phenylephrine, but not to carbachol, were significantly increased in PNL rats. The levels of NGF protein and mRNA in the bladder of PNL rats were significantly increased compared with sham rats. These results suggest that pudendal nerve neuropathy induced by PNL may be one of the potential risk factors for OAB, as well as SUI. Somato-visceral cross sensitization between somatic (pudendal) and visceral (bladder) sensory pathways that increases NGF expression and alpha(1)-adrenoceptor-mediated contractility in the bladder may be involved in this pathophysiological mechanism.     

Reactivity of aflatoxin B2a antibody with aflatoxin B1-modified DNA and related metabolites.

Aflatoxin B2a (AFB2a) antiserum has been previously used in an enzyme-linked immunosorbent assay (ELISA) for the quantitation of AFB1 and AFB2a. The present investigation examined the reactivity of the antiserum toward those adducts and metabolites of AFB1 believed to play a major role in aflatoxicosis and carcinogenesis. 2,3-Dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 (AFB1-N7-Gua), the putative 2,3-(N5-formyl-2-2', 5',6'-triamino-4-oxo-N5-pyrimidyl)-3-hydroxyaflatoxin B1 (AFB1-FAPyr), 2,3-dihydro-2,3-dihydroxyaflatoxin B1 (AFB1-diol), AFB1-N7-Gua-modified DNA, and AFB1-FAPyr-modified DNA were prepared by in vitro incubation or chemical methods and subjected to competitive AFB2a ELISA. The antiserum showed significant reactivity with all five compounds, indicating that it had a high degree of specificity for both the cyclopentenone and the methoxy group of the parent aflatoxin molecule. Sensitivity for AFB-N7-Gua-modified DNA, AFB1-FAPyr-modified DNA, and AFB1-diol by the ELISA method was 0.1 pmol per assay. To test the applicability of immunological detection of covalent binding of AFB1 to DNA, the ELISA was compared with a conventional radioisotopic assay in two in vitro studies. The results showed that estimates of the kinetics and substrate dependence of covalent binding to calf thymus DNA in rat microsomal incubation mixtures by both methods were comparable. The broad specificity AFB2a antibody might be of considerable value in the detection of AFB1 macromolecular adducts and related metabolites in epidemiological investigations or in the diagnosis of aflatoxicosis.     

A quantitative real-time PCR method for in planta monitoring of Phytophthora infestans growth

Aim: To develop a real‐time PCR‐based strategy for the detection of Paenibacillus larvae vegetative cells and spores to improve the diagnosis and the screening of American foulbrood (AFB), the most harmful pathology of honeybee brood. Methods and Results: A real‐time PCR that allowed selective identification and quantification of P. larvae 16S rRNA sequence was developed. Using standard samples quantified by flow cytometry, detection limits of 37·5 vegetative cells ml^?1 and 10 spores ml^?1 were determined. Compared to spread plate method, this real‐time PCR‐based strategy allowed, in only 2 h, the detection of P. larvae in contaminated honeys. No false‐positive results were obtained. Moreover, its detection limit was 100 times lower than that of the culture method (2 vs 200 spores g^?1 of honey). Conclusion: A rapid, selective, with low detection limit, sensitive and specific method to detect and quantify vegetative cells and spores of P. larvae is now available. Significance and Impact of Study: In addition to honey samples, this real‐time PCR‐based strategy may be also applied to confirm AFB diagnosis in honeybee brood and to screen other apiary supplies and products (bees, pollen, wax), thus broadening the control of AFB spreading.     

A new diagnostic tool for rapid and accurate detection of Mycobacterium tuberculosis

Mycobacterium tuberculosis, acid fast bacilli from the family of Mycobacteriaceae, is the causative agent of most cases of tuberculosis. Tuberculosis, as a communicable disease, remains a serious public health threat, killing more than one million people globally every year. Primary diagnosis of tuberculosis bacilli (TB) relies mainly on microscopic detection of acid fast bacilli (AFB), but the method suffers from low sensitivity and the results largely depend on the technician’s skill. New diagnostic tools are necessary to be introduced for rapid and accurate detection of the bacilli in sputum samples. We, in collaboration with Anda Biologicals, have developed a new platform, named as “Patho-tb”, for rapid detection of AFB with high sensitivity and with low dependence on human skills. Evaluation of Patho-tb test performance was done in two settings: (1) primary field study conducted using 38 sputa from high TB prevalence area of Iran (Zabol city near to the Afghanistan border), and (2) main study conducted using 476 sputa from Tehran, capital of Iran. Patho-tb was applied for processed sputum samples in parallel with routine diagnostic methods (including AFB microscopy, culture and PCR). All test results were compared to final clinical diagnostic state of an individual and diagnostic sensitivity (DSe), specificity, positive predictive value, negative predictive value and accuracy of each test results were calculated using standard formulations. Analytical sensitivity and specificity of the Patho-tb test were also determined. Calculated values for five above mentioned parameters are as follows: for field study: AFB (DSe: 29.6, DSp: 81.8, PPV: 80, NPV: 23.1, AC: 44.7), Patho-tb (DSe: 63, DSp: 72.7, PPV: 85, NPV: 44.4, AC: 65.8), and for main study: AFB (DSe: 86.1, DSp: 99.4, PPV: 98.5, NPV: 93.9, AC: 95.2), Patho-tb (DSe: 97.4, DSp: 92.9, PPV: 86.5, NPV: 98.7, AC: 94.3). Reproducibility of Patho-tb test results were near to 100% (Cohen’s kappa value between 0.85 and 1). The detection limit of Patho-tb test with 100% positivity rate was 3 × 10³ cells/ml of sputum. In the field study, Patho-tb test was 33.4% more sensitive than AFB microscopy, while the improvement was only 11.3% during the main study. Patho-tb results are easy to interpret and the test can be merged with other screening tests, like AFB. Totally, Patho-tb test alone or in conjunction with AFB microscopy is a useful screening tool for TB detection especially in poor geographical lab conditions.     

Boron exhibits hepatoprotective effect together with antioxidant,anti-inflammatory,and anti-apoptotic pathways in rats exposed to aflatoxin B1

BackgroundAflatoxins are one of the important environmental factors that pose a risk to living organisms. On the other hand, it has been indicated in research that boron intake has beneficial effects on organisms. In this study, the effect of boron was disclosed in rats exposed to aflatoxin B1 (AFB1), which poses a toxicological risk.MethodsA total of 36 male Sprague Dawley rats were separated into 6 groups and 0.125 mg/kg bw AFB1 and 5, 10, or 20 mg/kg bw doses of boron were given orally for 21 days. End of the experiment, biochemical, molecular, and histopathological analyses were performed.ResultsAFB1 treatment increased liver enzyme activities (AST, ALT, and ALP) and malondialdehyde level; on the other hand, it caused a decrease in glutathione level, superoxide dismutase and catalase activities. In addition, the mRNA expression levels of apoptotic (Bax, Caspase-3, Caspase-8, Caspase-9, and p53) and pro-inflammatory (TNF-α and NFκB) genes increased and the mRNA expression of the anti-apoptotic gene (Bcl-2) decreased in liver tissue. Also, AFB1 treatment increased DNA damage and caused histopathological alterations in the liver tissue. Additionally, boron applications at doses of 5, 10, and 20 mg/kg bw given with AFB1 reversed these negative changes.ConclusionsAs a result, boron exhibited hepatoprotective effect together with antioxidant, anti-inflammatory, and anti-apoptotic effects against AFB1-induced liver damage.     

Factors associated with negative direct sputum examination in Asian and African HIV-infected patients with tuberculosis (ANRS 1260)

Objective

To identify factors associated with negative direct sputum examination among African and Cambodian patients co-infected by Mycobacterium tuberculosis and HIV.

Design

Prospective multicenter study (ANRS1260) conducted in Cambodia, Senegal and Central African Republic.

Methods

Univariate and multivariate analyses (logistic regression) were used to identify clinical and radiological features associated with negative direct sputum examination in HIV-infected patients with positive M. tuberculosis culture on Lowenstein-Jensen medium.

Results

Between September 2002 and December 2005, 175 co-infected patients were hospitalized with at least one respiratory symptom and pulmonary radiographic anomaly. Acid-fast bacillus (AFB) examination was positive in sputum samples from 110 subjects (63%) and negative in 65 patients (37%). Most patients were at an advanced stage of HIV disease (92% at stage III or IV of the WHO classification) with a median CD4 cell count of 36/mm³. In this context, we found that sputum AFB negativity was more frequent in co-infected subjects with associated respiratory tract infections (OR = 2.8 [95%CI:1.1–7.0]), dyspnea (OR = 2.5 [95%CI:1.1–5.6]), and localized interstitial opacities (OR = 3.1 [95%CI:1.3–7.6]), but was less frequent with CD4≤50/mm³ (OR = 0.4 [95%CI:0.2–0.90), adenopathies (OR = 0.4 [95%CI:0.2–0.93]) and cavitation (OR = 0.1 [95%CI:0.03–0.6]).

Conclusions

One novel finding of this study is the association between concomitant respiratory tract infection and negative sputum AFB, particularly in Cambodia. This finding suggests that repeating AFB testing in AFB-negative patients should be conducted when broad spectrum antibiotic treatment does not lead to complete recovery from respiratory symptoms. In HIV-infected patients with a CD4 cell count below 50/mm3 without an identified cause of pneumonia, systematic AFB direct sputum examination is justified because of atypical clinical features (without cavitation) and high pulmonary mycobacterial burden.     

Leishmania (V.) braziliensis: detection by PCR in biopsies from patients with cutaneous leishmaniasis

Cutaneous leishmaniases present similar clinical appearances, but differing prognosis in the course of infection. Ulcers caused by parasites of the subgenus Viannia are more aggressive than ulcers caused by parasites of the subgenus Leishmania. Another problem is distinguishing between true Leishmania infection and other skin diseases in endemic areas, where cutaneous lesions and a single positive Montenegro intradermal test are enough to submit patients to specific treatment for cutaneous leishmaniasis. This study evaluated the efficacy of PCR in detecting in Leishmania in patients with cutaneous lesions. Leishmania (V.) braziliensis complex was determined by a primer pair from the multicopy spliced leader RNA. The results were compared to those of traditional methods. We analyzed biopsies of 109 patients with cutaneous lesions in the second most endemic region of Sao Paulo State, Brazil. Definitive diagnosis was established by clinical and “consensus laboratory criteria” (positive culture, stained tissue smears or PCR). Of 52 patients with cutaneous leishmaniasis, 96% had positive PCR, 69%, positive parasitological tests and 100%, positive Montenegro intradermal tests. Histopathological examination (only in 32 samples) were positive in 14 samples, suggestive in 14 and negative in 4 samples. All 57 patients with other etiologies had negative results in parasitological methods, PCR and histopathological examination (in 39 samples), but Montenegro intradermal tests were positive in 35%. PCR was highly sensitive and specific for L. (V.) braziliensis complex detection compared with other laboratory methods. Despite the specificity of the parasitological tests, the sensitivity was less than 70%. Montenegro intradermal reaction was highly sensitive, but with low specificity, only 65%. As suggestive results in histopathological examinations were shown in 14 samples, it was difficult to determine the true result. PCR applied to biopsies proved to be useful for differential diagnosis of cutaneous lesions of other etiologies in patients living in endemic areas. The advantages are most striking in clinical specimens with scarce amastigotes for which conventional methods have low sensitivity and should be considered for clinical and epidemiological patterns. On the other hand, both Montenegro intradermal test and parasitological methods are only modestly effective in cutaneous leishmaniasis diagnosis.     

Electrochemical immunosensor array using a 96-well screen-printed microplate for aflatoxin B1 detection

A novel analytical immunosensor array, based on a microtiter plate coupled to a multichannel electrochemical detection (MED) system using the intermittent pulse amperometry (IPA) technique, is proposed for the detection of aflatoxin B1 (AFB1). In the present work, the electrochemical behaviour and electroanalytical performance of the thick-film carbon sensors (also designated as screen-printed electrodes) incorporated in the multichannel electrochemical plate were first evaluated. Then the 96-well screen-printed microplate was modified in accord with a competitive indirect enzyme-linked immunoassay (ELISA) format for aflatoxin B1 detection. The measurements were performed using both spectrophotometric and electrochemical procedures and the results of the calibration curves, detection limit (LOD), sensitivity and reproducibility of the respective assay systems were evaluated. The immunoassay was then applied for analysis of corn samples spiked with AFB1 before and after the extraction treatment, in order to study the extraction efficiency and the matrix effect, respectively. These studies have shown that using this system, AFB1 can be measured at a level of 30 pg/mL and with a working range between 0.05 and 2 ng/mL. Good recoveries (103+/-8%) were obtained, demonstrating the suitability of the proposed assay for accurate determination of the AFB1 concentration in corn samples. The specificity of the assay was assessed by studying the cross-reactivity of PAb relative to AFB1. The results indicated that the PAb could readily distinguish AFB1 from other aflatoxins, with the exception for AFG1.     

Application of Genotype MTBDRplus in rapid detection of the Mycobacterium tuberculosis complex as well as its resistance to isoniazid and rifampin in a high volum laboratory in Southern China

The alarmingly worsening epidemics of drug-resistant tuberculosis (TB) call urgent need for a simple method for the rapid detection of drug-resistant TB in clinical settings. In an attempt to establish a rapid procedure for laboratory diagnosis of TB and investigate the local TB epidemiology, molecular line probe assay of the Genotype MTBDRplus was used to identify Mycobacterium tuberculosis complex (MTBC) and detect mutations conferring resistance to two most active first-line drugs against TB: Rifampin and Isoniazid. 96 acid-fast bacillus (AFB) smear- positive sputums and 18 PCR-positive non-sputum specimens have been determined for the MTBC and resistance to Rifampin and Isoniazid. The MTBC detection rates in two sources of specimens were 93.8% (90/96) and 77.8% (14/18) respectively. The overall drug resistance (Rifampin or Isoniazid) occurred in 34.6% (36/104). Resistance to rifampin (RMP) was 28.8% (30/104) and 25% (26/104) was to Isoniazid (INH), in which high level drug resistance accounted for 88.5% (23/26) and low level drug resistance accounted for 7.7% (2/26). Multidrug resistance (MDR), defined as resistant to both RMP and INH, was found in 19.2% (20/104) of clinical samples, which was double that of official statistics. In addition, 63.3% (19/30) RMP-resistant mutations were identified in the region of RopB 530–533 and 57.9% (11/19) were the S531L mutation. 84.6% (22/26) of resistance to INH was mediated by Kat S315T1 mutations which conferred the high-level resistance to INH. The Genotype MTBDRplus line probe assay is a suitable and applicable method for establishing the rapidness in detection of drug-resistant TB in clinical laboratory. It will be a valuable addition to the conventional TB diagnostic approaches.