Divergent Profile of Emerging Cutaneous Leishmaniasis in Subtropical Brazil: New Endemic Areas in the Southern Frontier

Background

Although known to be highly endemic in the Amazon regions of Brazil, the presence of cutaneous leishmaniasis (CL) in the subtropical southern part of the country has largely been ignored. This study was conducted to demonstrate CL is emerging in the Brazilian state of Santa Catarina, as well as to characterize the epidemiological profile and Leishmania species involved.

Methodology/Principal Findings

For this cross-sectional study, data from all CL cases from Santa Catarina, Brazil, reported to the Brazilian National Notifiable Diseases Information System from 2001 to 2009 were investigated. Amplification of the kDNA minicircle conserved region followed by restriction fragment length polymorphism (PCR-RFLP) was conducted to screen for Leishmania species present in patient biopsy. Overall, 542 CL cases were reported, with majority resulting from autochthonous transmission (n = 401, 73.99%) and occurring in urban zones (n = 422, 77.86%). Age, gender, zone of residence, origin of case, clinical form and case outcome were found to differ significantly by region. Imported cases were over seven times more likely to relapse (95% CI 2.56–21.09). Mapping of cases revealed new endemic areas in northeastern Santa Catarina with two species present. With the exception of three L. (Leishmania) amazonensis cases (1.20%), majority of PCR positive samples were found to be L. (Viannia) braziliensis (n = 248, 98.80%).

Conclusions/Significance

CL is now endemic in the state of Santa Catarina, Brazil, with case profiles varying significantly by region. L. (V.) braziliensis has been identified as the predominant species in the region.     

Frequency of Chromosomally-Integrated Human Herpesvirus 6 in Children with Acute Lymphoblastic Leukemia

Introduction

Human herpesvirus 6 (HHV-6) is a ubiquitous pathogen infecting nearly 100% of the human population. Of these individuals, between 0.2% and 1% of them carry chromosomally-integrated HHV-6 (ciHHV-6). The biological consequences of chromosomal integration by HHV-6 remain unknown.

Objective

To determine and compare the frequency of ciHHV-6 in children with acute lymphoblastic leukemia to healthy blood donors.

Methodology

A total of 293 DNA samples from children with pre-B (n = 255), pre-pre-B (n = 4), pre-T (n = 26) and undetermined (n = 8) leukemia were analyzed for ciHHV-6 by quantitative TaqMan PCR (QPCR) using HHV-6 specific primers and probe. As control, DNA samples from 288 healthy individuals were used. Primers and probe specific to the cellular GAPDH gene were used to estimate integrity and DNA content.

Results

Out of 293 DNA samples from the leukemic cohort, 287 contained amplifiable DNA. Of these, only 1 (0.35%) contained ciHHV-6. Variant typing indicates that the ci-HHV-6 corresponds to variant A. None of the 288 DNA samples from healthy individuals contained ciHHV-6.

Conclusion

The frequency of ciHHV-6 in children with acute lymphoblastic leukemia is similar (p = 0.5) to that of healthy individuals. These results suggest that acute lymphoblastic leukemia does not originate as a consequence to integration of HHV-6 within the chromosomes.     

Cytokine Responses to Novel Antigens in an Indian Population Living in an Area Endemic for Visceral Leishmaniasis

Background

There are no effective vaccines for visceral leishmaniasis (VL), a neglected parasitic disease second only to malaria in global mortality. We previously identified 14 protective candidates in a screen of 100 Leishmania antigens as DNA vaccines in mice. Here we employ whole blood assays to evaluate human cytokine responses to 11 of these antigens, in comparison to known defined and crude antigen preparations.

Methods

Whole blood assays were employed to measure IFN-γ, TNF-α and IL-10 responses to peptide pools of the novel antigens R71, Q51, L37, N52, L302.06, J89, M18, J41, M22, M63, M57, as well as to recombinant proteins of tryparedoxin peroxidase (TRYP), Leishmania homolog of the receptor for activated C kinase (LACK) and to crude soluble Leishmania antigen (SLA), in Indian patients with active (n = 8) or cured (n = 16) VL, and in modified Quantiferon positive (EHC^+ve, n = 20) or modified Quantiferon negative (EHC^−ve, n = 9) endemic healthy controls (EHC).

Results

Active VL, cured VL and EHC^+ve groups showed elevated SLA-specific IFN-γ, but only active VL patients produced IL-10 and EHC^+ve did not make TNF-α. IFN-γ to IL-10 and TNF-α to IL-10 ratios in response to TRYP and LACK antigens were higher in cured VL and EHC^+ve exposed individuals compared to active VL. Five of the eleven novel candidates (R71, L37, N52, J41, and M22) elicited IFN-γ and TNF-α, but not IL-10, responses in cured VL (55–87.5% responders) and EHC^+ve (40–65% responders) subjects.

Conclusions

Our results are consistent with an important balance between pro-inflammatory IFNγ and TNFγ cytokine responses and anti-inflammatory IL-10 in determining outcome of VL in India, as highlighted by response to both crude and defined protein antigens. Importantly, cured VL patients and endemic Quantiferon positive individuals recognise 5 novel vaccine candidate antigens, confirming our recent data for L. chagasi in Brazil, and their potential as cross-species vaccine candidates.     

Canine skin and conjunctival swab samples for the detection and quantification of Leishmania infantum DNA in an endemic urban area in Brazil

Background

We evaluated kDNA PCR/hybridization and quantitative real-time PCR (qPCR) targeting the gene of DNA polymerase of Leishmania infantum for CVL diagnosis and assessment of parasite load in clinical samples obtained invasively and non-invasively.

Methodology/Principal Findings

Eighty naturally infected dogs from an endemic urban area in Brazil were used. Animals were divided into two groups based on the presence or absence of CVL clinical sings. Skin biopsies, bone marrow, blood and conjunctival swabs samples were collected and submitted to L. infantum DNA detection. In addition, anti-Leishmania antibody titers were measured by Immunofluorescence antibody test. The symptomatic dogs had increased titers compared to asymptomatic dogs (P = 0.025). The frequencies of positive results obtained by kDNA PCR/hybridization for asymptomatic and symptomatic dogs, respectively, were as follows: right conjunctiva, 77.5% and 95.0%; left conjunctiva, 75.0% and 87.5%; skin, 45.0% and 75.0%; bone marrow, 50.0% and 77.5%; and blood, 27.5% and 22.5%. In both groups, the parasite load in the skin samples was the highest (P<0.0001). The parasite loads in the conjunctival swab and bone marrow samples were statistically equivalent within each group. The parasite burden in conjunctival swabs was higher in the dogs with clinical signs than in asymptomatic dogs (P = 0.028). This same relationship was also observed in the bone marrow samples (P = 0.002). No differences in amastigotes load in the skin were detected between the groups.

Conclusions

The conjunctival swab is a suitable clinical sample for qualitative molecular diagnosis of CVL. The highest parasite burdens were detected in skin regardless of the presence of VL-associated clinical signs. The qPCR results emphasized the role of dogs, particularly asymptomatic dogs, as reservoirs for CVL because of the high cutaneous parasite loads. These results may help to explain the maintenance of high transmission rates and numbers of CVL cases in endemic urban regions.     

Iron Status and Helicobacter pylori Infection in Symptomatic Children: An International Multi-Centered Study

Objective

Iron deficiency (ID) and iron deficiency anaemia (IDA) are global major public health problems, particularly in developing countries. Whilst an association between H. pylori infection and ID/IDA has been proposed in the literature, currently there is no consensus. We studied the effects of H. pylori infection on ID/IDA in a cohort of children undergoing upper gastrointestinal endoscopy for upper abdominal pain in two developing and one developed country.

Methods

In total 311 children (mean age 10.7±3.2 years) from Latin America - Belo Horizonte/Brazil (n = 125), Santiago/Chile (n = 105) - and London/UK (n = 81), were studied. Gastric and duodenal biopsies were obtained for evaluation of histology and H. pylori status and blood samples for parameters of ID/IDA.

Results

The prevalence of H. pylori infection was 27.7% being significantly higher (p<0.001) in Latin America (35%) than in UK (7%). Multiple linear regression models revealed H. pylori infection as a significant predictor of low ferritin and haemoglobin concentrations in children from Latin-America. A negative correlation was observed between MCV (r = −0.26; p = 0.01) and MCH (r = −0.27; p = 0.01) values and the degree of antral chronic inflammation, and between MCH and the degree of corpus chronic (r = −0.29, p = 0.008) and active (r = −0.27, p = 0.002) inflammation.

Conclusions

This study demonstrates that H. pylori infection in children influences the serum ferritin and haemoglobin concentrations, markers of early depletion of iron stores and anaemia respectively.     

Cranialization of the Frontal Sinus for Secondary Mucocele Prevention following Open Surgery for Benign Frontal Lesions

Objective

To compare frontal sinus cranialization to obliteration for future prevention of secondary mucocele formation following open surgery for benign lesions of the frontal sinus.

Study Design

Retrospective case series.

Setting

Tertiary academic medical center.

Patients

Sixty-nine patients operated for benign frontal sinus pathology between 1994 and 2011.

Interventions

Open excision of benign frontal sinus pathology followed by either frontal obliteration (n = 41, 59%) or frontal cranialization (n = 28, 41%).

Main Outcome Measures

The prevalence of post-surgical complications and secondary mucocele formation were compiled.

Results

Pathologies included osteoma (n = 34, 49%), mucocele (n = 27, 39%), fibrous dysplasia (n = 6, 9%), and encephalocele (n = 2, 3%). Complications included skin infections (n = 6), postoperative cutaneous fistula (n = 1), telecanthus (n = 4), diplopia (n = 3), nasal deformity (n = 2) and epiphora (n = 1). None of the patients suffered from postoperative CSF leak, meningitis or pneumocephalus. Six patients, all of whom had previously undergone frontal sinus obliteration, required revision surgery due to secondary mucocele formation. Statistical analysis using non-inferiority test reveal that cranialization of the frontal sinus is non-inferior to obliteration for preventing secondary mucocele formation (P<0.0001).

Conclusion

Cranialization of the frontal sinus appears to be a good option for prevention of secondary mucocele development after open excision of benign frontal sinus lesions.     

Epidemiologic aspects of an emerging focus of visceral leishmaniasis in Tbilisi, Georgia

Background

Over the last 15 years, visceral leishmaniasis (VL) has emerged as a public health concern in Tbilisi, the capital of Georgia.

Methodology/Principal Findings

Seroepidemiological surveys were conducted to determine the prevalence and incidence of infection in children and dogs within the main focus of VL, and to identify risk factors associated with human infection. Of 4,250 children investigated, 7.3% were positive by direct agglutination test in a baseline survey; an apparent incidence rate of 6.0% was estimated by one year follow-up. None of the seropositive children progressed to VL during the survey. Increased seropositivity at one year was predicted by presence at baseline of clustered flying insects (OR = 1.49; P = 0.001), perceived satisfactory sanitation (OR = 1.65; P<0.001), stray dogs (OR = 1.33; P = 0.023), and by persistent fever during the 6 months prior to baseline survey (OR = 14.2; P<0.001). Overall, 18.2% (107/588) of domestic and 15.3% (110/718) of stray dogs were seropositive by the rk39 dipstick test. Clinical VL signs were found in 1.3% of domestic and 2.9% of stray, seropositive dogs. Parasites isolated from human and dog samples were identified by PCR and phylogenetic analysis of the Leishmania 70 kDa heat-shock protein (HSP70) gene as Leishmania infantum.

Conclusions/Significance

There is an active focus of L. infantum transmission in Tbilisi with a high prevalence of human and canine infections.     

Clinical Utility of the Cryptococcal Antigen Lateral Flow Assay in a Diagnostic Mycology Laboratory

Background

Cryptococcus neoformans causes life-threatening meningitis. A recently introduced lateral flow immunoassay (LFA) to detect cryptococcal antigen (CRAG) is reportedly more rapid and convenient than standard latex agglutination (LA), but has not yet been evaluated in a diagnostic laboratory setting.

Methods

One hundred and six serum, 42 cerebrospinal fluid (CSF), and 20 urine samples from 92 patients with known or suspected cryptococcosis were tested by LA and LFA, and titres were compared. Results were correlated with laboratory-confirmed cryptococcosis. Serial samples were tested in nine treated patients.

Results

Twenty-five of 92 patients had confirmed cryptococcosis; all sera (n = 56) from these patients were positive by LFA (sensitivity 100%, 95% confidence interval (CI) 93.6–100%) compared with 51/56 positive by LA (sensitivity 91.1%, 95% CI 80.7–96.1%). Fifty sera from 67 patients without cryptococcosis tested negative in both assays. While LA yielded more false negative results (5/56) this did not reach statistical significance (p = 0.063). Nine CSF samples from patients with cryptococcal meningitis yielded positive results using both assays while 17/18 urine samples from patients with cryptococcosis were positive by the LFA. The LFA detected CRAG in C. gattii infection (n = 4 patients). Agreement between titres obtained by both methods (n = 38 samples) was imperfect; correlation between log-transformed titres (r) was 0.84. Turn-around-time was 20 minutes for the LFA and 2 h for LA. The cost per qualitative sample was 18USD and 91 USD, respectively and per quantitative sample was 38USD and 144USD, respectively.

Conclusions

Qualitative agreement between the LFA and LA assays performed on serum and CSF was good but agreement between titres was imperfect. Ease of performance of the LFA and the capacity for testing urine suggest it has a role in the routine laboratory as a rapid diagnostic test or point-of-care test.     

Cerebrospinal Fluid Interleukin-6 in Central Nervous System Inflammatory Diseases

Background

Interleukin (IL)-6 is recognised as an important cytokine involved in inflammatory diseases of the central nervous system (CNS).

Objective

To perform a large retrospective study designed to test cerebrospinal fluid (CSF) IL-6 levels in the context of neurological diseases, and evaluate its usefulness as a biomarker to help discriminate multiple sclerosis (MS) from other inflammatory neurological diseases (OIND).

Patients and Methods

We analyzed 374 CSF samples for IL-6 using a quantitative enzyme-linked immunosorbent assay. Groups tested were composed of demyelinating diseases of the CNS (DD, n = 117), including relapsing-remitting MS (RRMS, n = 65), primary progressive MS (PPMS, n = 11), clinically isolated syndrome (CIS, n = 11), optic neuritis (ON, n = 30); idiopathic transverse myelitis (ITM, n = 10); other inflammatory neurological diseases (OIND, n = 35); and non-inflammatory neurological diseases (NIND, n = 212). Differences between groups were analysed using Kruskal−Wallis test and Mann−Whitney U-test.

Results

CSF IL-6 levels exceeded the positivity cut-off of 10 pg/ml in 18 (51.4%) of the 35 OIND samples, but in only three (3.9%) of the 76 MS samples collected. CSF IL-6 was negative for all NIND samples tested (0/212). IL-6 cut-off of 10 pg/ml offers 96% sensitivity to exclude MS.

Conclusion

CSF IL-6 may help to differentiate MS from its major differential diagnosis group, OIND.     

Comparison of PfHRP-2/pLDH ELISA,qPCR and Microscopy for the Detection of Plasmodium Events and Prediction of Sick Visits during a Malaria Vaccine Study

Background

Compared to expert malaria microscopy, malaria biomarkers such as Plasmodium falciparum histidine rich protein-2 (PfHRP-2), and PCR provide superior analytical sensitivity and specificity for quantifying malaria parasites infections. This study reports on parasite prevalence, sick visits parasite density and species composition by different diagnostic methods during a phase-I malaria vaccine trial.

Methods

Blood samples for microscopy, PfHRP-2 and Plasmodium lactate dehydrogenase (pLDH) ELISAs and real time quantitative PCR (qPCR) were collected during scheduled (n = 298) or sick visits (n = 38) from 30 adults participating in a 112-day vaccine trial. The four methods were used to assess parasite prevalence, as well as parasite density over a 42-day period for patients with clinical episodes.

Results

During scheduled visits, qPCR (39.9%, N = 119) and PfHRP-2 ELISA (36.9%, N = 110) detected higher parasite prevalence than pLDH ELISA (16.8%, N = 50) and all methods were more sensitive than microscopy (13.4%, N = 40). All microscopically detected infections contained P. falciparum, as mono-infections (95%) or with P. malariae (5%). By qPCR, 102/119 infections were speciated. P. falciparum predominated either as monoinfections (71.6%), with P. malariae (8.8%), P. ovale (4.9%) or both (3.9%). P. malariae (6.9%) and P. ovale (1.0%) also occurred as co-infections (2.9%). As expected, higher prevalences were detected during sick visits, with prevalences of 65.8% (qPCR), 60.5% (PfHRP-2 ELISA), 21.1% (pLDH ELISA) and 31.6% (microscopy). PfHRP-2 showed biomass build-up that climaxed (1813±3410 ng/mL SD) at clinical episodes.

Conclusion

PfHRP-2 ELISA and qPCR may be needed for accurately quantifying the malaria parasite burden. In addition, qPCR improves parasite speciation, whilst PfHRP-2 ELISA is a potential predictor for clinical disease caused by P. falciparum.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00666380","term_id":"NCT00666380"}}NCT00666380     

The Prognostic Role of Ezrin Immunoexpression in Osteosarcoma: A Meta-Analysis of Published Data

Background

The significance of ezrin immunoexpression and prognosis for osteosarcoma is still controversial. The aim was to provide a meta-analysis for ezrin immunoexpression and prognostic features of osteosarcoma patients.

Methods

A detailed search was made in MEDLINE, EMBASE and the Web of Knowledge for relevant original articles published in English; methodological quality of the included studies was also assessed. Two reviewers extracted data independently. Studies were pooled and summary hazard ratios (HRs) and odds ratio (ORs) with corresponding confidence intervals (CIs) were calculated.

Results

Final analysis of 318 patients from 5 eligible studies was performed. Combined HR of ezrin immunohistochemical staining suggested that positive immunoexpression had an unfavorable impact on osteosarcoma patients'' overall survival (n = 223 in 4 studies; HR = 4.79; 95% CI: 1.50–15.30; P = 0.008) but not on event-free survival (n = 202 in 3 studies; HR = 1.59; 95% CI: 0.61–4.15; P = 0. 0.342). Combined OR of ezrin immunohistochemical staining indicated that positive immunoexpression was associated with recurrence (n = 134 in 2 studies; OR = 3.79; 95% CI: 1.49–9.64; P = 0.005) but not with serum ALP level (n = 160 in 2 studies; OR = 2.16; 95% CI: 0.09–52.50; P = 0.637) and histological response to neoadjuvant chemotherapy(n = 260 in 4 studies; OR = 0.87; 95% CI: 0.37–2.03; P = 0.740).

Conclusions

The results of this meta-analysis suggest that ezrin positive immunoexpression confers a higher risk of recurrence and a worse survival in osteosarcoma patients. Large prospective studies are needed to provide solid data to investigate the precise prognostic significance of ezrin.     

Etiologic Agents of Central Nervous System Infections among Febrile Hospitalized Patients in the Country of Georgia

Objectives

There is a large spectrum of viral, bacterial, fungal, and prion pathogens that cause central nervous system (CNS) infections. As such, identification of the etiological agent requires multiple laboratory tests and accurate diagnosis requires clinical and epidemiological information. This hospital-based study aimed to determine the main causes of acute meningitis and encephalitis and enhance laboratory capacity for CNS infection diagnosis.

Methods

Children and adults patients clinically diagnosed with meningitis or encephalitis were enrolled at four reference health centers. Cerebrospinal fluid (CSF) was collected for bacterial culture, and in-house and multiplex RT-PCR testing was conducted for herpes simplex virus (HSV) types 1 and 2, mumps virus, enterovirus, varicella zoster virus (VZV), Streptococcus pneumoniae, HiB and Neisseria meningitidis.

Results

Out of 140 enrolled patients, the mean age was 23.9 years, and 58% were children. Bacterial or viral etiologies were determined in 51% of patients. Five Streptococcus pneumoniae cultures were isolated from CSF. Based on in-house PCR analysis, 25 patients were positive for S. pneumoniae, 6 for N. meningitidis, and 1 for H. influenzae. Viral multiplex PCR identified infections with enterovirus (n = 26), VZV (n = 4), and HSV-1 (n = 2). No patient was positive for mumps or HSV-2.

Conclusions

Study findings indicate that S. pneumoniae and enteroviruses are the main etiologies in this patient cohort. The utility of molecular diagnostics for pathogen identification combined with the knowledge provided by the investigation may improve health outcomes of CNS infection cases in Georgia.     

A Schistosoma haematobium-Specific Real-Time PCR for Diagnosis of Urogenital Schistosomiasis in Serum Samples of International Travelers and Migrants

Background

Diagnosis of urogenital schistosomiasis by microscopy and serological tests may be elusive in travelers due to low egg load and the absence of seroconversion upon arrival. There is need for a more sensitive diagnostic test. Therefore, we developed a real-time PCR targeting the Schistosoma haematobium-specific Dra1 sequence.

Methodology/Principal Findings

The PCR was evaluated on urine (n = 111), stool (n = 84) and serum samples (n = 135), and one biopsy from travelers and migrants with confirmed or suspected schistosomiasis. PCR revealed a positive result in 7/7 urine samples, 11/11 stool samples and 1/1 biopsy containing S. haematobium eggs as demonstrated by microscopy and in 22/23 serum samples from patients with a parasitological confirmed S. haematobium infection. S. haematobium DNA was additionally detected by PCR in 7 urine, 3 stool and 5 serum samples of patients suspected of having schistosomiasis without egg excretion in urine and feces. None of these suspected patients demonstrated other parasitic infections except one with Blastocystis hominis and Entamoeba cyst in a fecal sample. The PCR was negative in all stool samples containing S. mansoni eggs (n = 21) and in all serum samples of patients with a microscopically confirmed S. mansoni (n = 22), Ascaris lumbricoides (n = 1), Ancylostomidae (n = 1), Strongyloides stercoralis (n = 1) or Trichuris trichuria infection (n = 1). The PCR demonstrated a high specificity, reproducibility and analytical sensitivity (0.5 eggs per gram of feces).

Conclusion/Significance

The real-time PCR targeting the Dra1 sequence for S. haematobium-specific detection in urine, feces, and particularly serum, is a promising tool to confirm the diagnosis, also during the acute phase of urogenital schistosomiasis.     

The Fecal Microbiome in Dogs with Acute Diarrhea and Idiopathic Inflammatory Bowel Disease

Background

Recent molecular studies have revealed a highly complex bacterial assembly in the canine intestinal tract. There is mounting evidence that microbes play an important role in the pathogenesis of acute and chronic enteropathies of dogs, including idiopathic inflammatory bowel disease (IBD). The aim of this study was to characterize the bacterial microbiota in dogs with various gastrointestinal disorders.

Methodology/Principal Findings

Fecal samples from healthy dogs (n = 32), dogs with acute non-hemorrhagic diarrhea (NHD; n = 12), dogs with acute hemorrhagic diarrhea (AHD; n = 13), and dogs with active (n = 9) and therapeutically controlled idiopathic IBD (n = 10) were analyzed by 454-pyrosequencing of the 16S rRNA gene and qPCR assays. Dogs with acute diarrhea, especially those with AHD, had the most profound alterations in their microbiome, as significant separations were observed on PCoA plots of unweighted Unifrac distances. Dogs with AHD had significant decreases in Blautia, Ruminococcaceae including Faecalibacterium, and Turicibacter spp., and significant increases in genus Sutterella and Clostridium perfringens when compared to healthy dogs. No significant separation on PCoA plots was observed for the dogs with IBD. Faecalibacterium spp. and Fusobacteria were, however, decreased in the dogs with clinically active IBD, but increased during time periods of clinically insignificant IBD, as defined by a clinical IBD activity index (CIBDAI).

Conclusions

Results of this study revealed a bacterial dysbiosis in fecal samples of dogs with various GI disorders. The observed changes in the microbiome differed between acute and chronic disease states. The bacterial groups that were commonly decreased during diarrhea are considered to be important short-chain fatty acid producers and may be important for canine intestinal health. Future studies should correlate these observed phylogenetic differences with functional changes in the intestinal microbiome of dogs with defined disease phenotypes.     

A Comparison between Droplet Digital and Quantitative PCR in the Analysis of Bacterial 16S Load in Lung Tissue Samples from Control and COPD GOLD 2

Background

Low biomass in the bacterial lung tissue microbiome utilizes quantitative PCR (qPCR) 16S bacterial assays at their limit of detection. New technology like droplet digital PCR (ddPCR) could allow for higher sensitivity and accuracy of quantification. These attributes are needed if specific bacteria within the bacterial lung tissue microbiome are to be evaluated as potential contributors to diseases such as chronic obstructive pulmonary disease (COPD). We hypothesize that ddPCR is better at quantifying the total bacterial load in lung tissue versus qPCR.

Methods

Control (n = 16) and COPD GOLD 2 (n = 16) tissue samples were obtained from patients who underwent lung resection surgery, were cut on a cryotome, and sections were assigned for use in quantitative histology or for DNA extraction. qPCR and ddPCR were performed on these samples using primers spanning the V2 region on the 16S rRNA gene along with negative controls. Total 16S counts were compared between the two methods. Both methods were assessed for correlations with quantitative histology measurements of the tissue.

Results

There was no difference in the average total 16S counts (P>0.05) between the two methods. However, the negative controls contained significantly lower counts in the ddPCR (0.55 ± 0.28 16S/uL) than in the qPCR assay (1.00 ± 0.70 16S copies) (P <0.05). The coefficient of variation was significantly lower for the ddPCR assay (0.18 ± 0.14) versus the qPCR assay (0.62 ± 0.29) (P<0.05).

Conclusion

Overall the ddPCR 16S assay performed better by reducing the background noise in 16S of the negative controls compared with 16S qPCR assay.     

Conjunctival MicroRNA Expression in Inflammatory Trachomatous Scarring

Purpose

Trachoma is a fibrotic disease of the conjunctiva initiated by Chlamydia trachomatis infection. This blinding disease affects over 40 million people worldwide yet the mechanisms underlying its pathogenesis remain poorly understood. We have investigated host microRNA (miR) expression in health (N) and disease (conjunctival scarring with (TSI) and without (TS) inflammation) to determine if these epigenetic differences are associated with pathology.

Methods

We collected two independent samples of human conjunctival swab specimens from individuals living in The Gambia (n = 63 & 194). miR was extracted, and we investigated the expression of 754 miR in the first sample of 63 specimens (23 N, 17 TS, 23 TSI) using Taqman qPCR array human miRNA genecards. Network and pathway analysis was performed on this dataset. Seven miR that were significantly differentially expressed between different phenotypic groups were then selected for validation by qPCR in the second sample of 194 specimens (93 N, 74 TS, 22 TSI).

Results

Array screening revealed differential expression of 82 miR between N, TS and TSI phenotypes (fold change >3, p<0.05). Predicted mRNA targets of these miR were enriched in pathways involved in fibrosis and epithelial cell differentiation. Two miR were confirmed as being differentially expressed upon validation by qPCR. miR-147b is significantly up-regulated in TSI versus N (fold change = 2.3, p = 0.03) and miR-1285 is up-regulated in TSI versus TS (fold change = 4.6, p = 0.005), which was consistent with the results of the qPCR array.

Conclusions

miR-147b and miR-1285 are up-regulated in inflammatory trachomatous scarring. Further investigation of the function of these miR will aid our understanding of the pathogenesis of trachoma.     

High Prevalence of Human Cytomegalovirus Proteins and Nucleic Acids in Primary Breast Cancer and Metastatic Sentinel Lymph Nodes

Background

Breast cancer is a leading cause of death among women worldwide. Increasing evidence implies that human cytomegalovirus (HCMV) infection is associated with several malignancies. We aimed to examine whether HCMV is present in breast cancer and sentinel lymph node (SLN) metastases.

Materials and Methods

Formalin-fixed paraffin-embedded tissue specimens from breast cancer and paired sentinel lymph node (SLN) samples were obtained from patients with (n = 35) and without SLN metastasis (n = 38). HCMV immediate early (IE) and late (LA) proteins were detected using a sensitive immunohistochemistry (IHC) technique and HCMV DNA by real-time PCR.

Results

HCMV IE and LA proteins were abundantly expressed in 100% of breast cancer specimens. In SLN specimens, 94% of samples with metastases (n = 34) were positive for HCMV IE and LA proteins, mostly confined to neoplastic cells while some inflammatory cells were HCMV positive in 60% of lymph nodes without metastases (n = 35). The presence of HCMV DNA was confirmed in 12/12 (100%) of breast cancer and 10/11 (91%) SLN specimens from the metastatic group, but was not detected in 5/5 HCMV-negative, SLN-negative specimens. There was no statistically significant association between HCMV infection grades and progesterone receptor, estrogen receptor alpha and Elston grade status.

Conclusions

The role of HCMV in the pathogenesis of breast cancer is unclear. As HCMV proteins were mainly confined to neoplastic cells in primary breast cancer and SLN samples, our observations raise the question whether HCMV contributes to the tumorigenesis of breast cancer and its metastases.     

Is IP-10 a Better Biomarker for Active and Latent Tuberculosis in Children than IFNγ?

Background

The blood based interferon-gamma release assays (IGRA) for the diagnosis of tuberculosis do not discriminate between active TB disease and latent TB infection (LTBI). The search for distinguishing biomarkers therefore continues, as the accurate diagnosis of tuberculosis is particularly challenging in children. IFN-γ-inducible protein 10 (IP-10/CXCL10) has recently been evaluated as a marker for active TB in adults with promising results.

Aim

To investigate this new biomarker for active TB and LTBI in paediatrics.

Method

We measured IP-10 levels using ELISA in supernatants of whole blood samples stimulated with TB-specific-antigens and negative control antigen.

Results

IP-10 is produced in high levels following mycobacterial antigen stimulation in active TB (n = 17) and LTBI (n = 16) compared to controls (n = 16) and to IFN-γ. The baseline levels of IP-10 are increased in active TB and in LTBI, but there is no significant difference of stimulated levels of IP-10 between active TB and LTBI.

Conclusions

IP-10 is a biomarker for tuberculosis in children. However like IFNγ, IP-10 also does not distinguish between active TB and LTBI.     

Inaccuracy of Enzyme-Linked Immunosorbent Assay Using Soluble and Recombinant Antigens to Detect Asymptomatic Infection by Leishmania infantum

Background

One of the most important drawbacks in visceral leishmaniasis (VL) population studies is the difficulty of diagnosing asymptomatic carriers. The aim of this study, conducted in an urban area in the Southeast of Brazil, was to evaluate the performance of serology to identify asymptomatic VL infection in participants selected from a cohort with a two-year follow-up period.

Methodology

Blood samples were collected in 2001 from 136 cohort participants (97 positive and 39 negatives, PCR/hybridization carried out in 1999). They were clinically evaluated and none had progressed to disease from their asymptomatic state. As controls, blood samples from 22 control individuals and 8 patients with kala-azar were collected. Two molecular biology techniques (reference tests) were performed: PCR with Leishmania-generic primer followed by hybridization using L. infantum probe, and PCR with specific primer to L. donovani complex. Plasma samples were tested by ELISA using three different antigens: L. infantum and L. amazonensis crude antigens, and rK39 recombinant protein. Accuracy of the serological tests was evaluated using sensitivity, specificity, likelihood ratio and ROC curve.

Findings

The presence of Leishmania was confirmed, by molecular techniques, in all kala-azar patients and in 117 (86%) of the 136 cohort participants. Kala-azar patients showed high reactivity in ELISAs, whereas asymptomatic individuals presented low reactivity against the antigens tested. When compared to molecular techniques, the L. amazonensis and L. infantum antigens showed higher sensitivity (49.6% and 41.0%, respectively) than rK39 (26.5%); however, the specificity of rK39 was higher (73.7%) than L. amazonensis (52.6%) and L. infantum antigens (36.8%). Moreover, there was low agreement among the different antigens used (kappa<0.10).

Conclusions

Serological tests were inaccurate for diagnosing asymptomatic infections compared to molecular methods; this could lead to misclassification bias in population studies. Therefore, studies which have used serological assays to estimate prevalence, to evaluate intervention programs or to identify risk factors for Leishmania infection, may have had their results compromised.     

Non-Tuberculous Mycobacteria and the Performance of Interferon Gamma Release Assays in Denmark

Background

The QuantiFERON-TB-Gold Test (QFT) is more specific than the Mantoux skin-test to discriminate between Mycobacterium tuberculosis (MTB) and non-tuberculous mycobacterial (NTM) infections. Here we study the performance of the QFT in patients with NTM disease.

Methods

From 2005 to 2011, nationwide patient data on positive NTM cultures (n = 925) were combined with nationwide data on QFT results (n = 16,133), both retrieved from the International Reference Laboratory of Mycobacteriology, Denmark. A total of 112 patients with NTM infections had a QFT performed, 53 patients had definite NTM disease, 10 had possible disease and 49 had NTM colonization.

Results

QFT was positive in 8% (4/53) of patients with definite disease, 40% (4/10) with possible disease and 31% (15/49) with colonization. Positivity rate was lowest among patients with definite disease infected with NTM without the RD1 region 4% (2/50). None of the 15 children with MAC lymphadenitis had a positive QFT.

Conclusion

This study is one of the largest assessing IGRAs in patients with NTM disease in a TB low-incidence setting. Our study showed that the QFT holds potential to discriminate between NTM and MTB infections. We found no positive IGRA test results among children with NTM not sharing the RD1-region of MTB resulting in a 100% specificity and we suggest that a QFT in a child presenting with cervical lymphadenitis may be helpful in distinguishing NTM from TB lymphadenitis.