Minimum detection limit of an in-house nested-PCR assay for herpes simplex virus and varicella zoster virus

Introduction Herpes simplex virus (HSV) and varicella zoster virus (VZV) are responsible for a variety of human diseases, including central nervous system diseases. The use of polymerase chain reaction (PCR) techniques on cerebrospinal fluid samples has allowed the detection of viral DNA with high sensitivity and specificity. Methods Serial dilutions of quantified commercial controls of each virus were subjected to an in-house nested-PCR technique. Results The minimum detection limits for HSV and VZV were 5 and 10 copies/µL, respectively. Conclusions The detection limit of nested-PCR for HSV and VZV in this study was similar to the limits found in previous studies.

Analysis of the detection coefficient for the association between leprosy and pregnancy in the integration region of Carajas, State of Para, Brazil

Introduction The association between leprosy and pregnancy is currently poorly understood and has been linked to serious clinical consequences. Methods A retrospective study between 2007 and 2009 was performed in the integration region of Carajás, Brazil on a population of pregnant lepers, with non-lepers of ages 12-49 years serving as the reference population. Results Twenty-nine pregnant lepers were studied during the study period. The detection rates (DRs) for the studied association were 4.7 in 2007, 9.4 in 2008, and 4.3 in 2009. Conclusions The Carajás region presented a medium pattern of endemicity during most of the study period, with a high DR found in 2008.

Detection of arboviruses of public health interest in free-living New World primates (Sapajus spp.; Alouatta caraya) captured in Mato Grosso do Sul, Brazil

Introduction A sero-epidemiological survey was undertaken to detect the circulation of arboviruses in free-living non-human primates. Methods Blood samples were obtained from 16 non-human primates (13 Sapajus spp. and three Alouatta caraya) that were captured using terrestrial traps and anesthetic darts in woodland regions in the municipalities of Campo Grande, Aquidauana, Jardim, Miranda and Corumbá in the State of Mato Grosso do Sul, Brazil. The samples were sent to the Instituto Evandro Chagas (IEC) in Ananindeua, Pará, Brazil, to detect antibodies against 19 species of arboviruses using a hemagglutination inhibition test (HI). Results Of the 16 primates investigated in the present study, five (31.2%) were serologically positive for an arbovirus. Of these five, two (12.5%) exhibited antibodies to the Flavivirus genus, one (6.2%) exhibited a monotypic reaction to Cacipacoré virus, one (6.2%) was associated with Mayaro virus, and one (6.2%) was positive for Oropouche virus. Conclusions Based on the positive serology observed in the present study, it was possible to conclude that arboviruses circulate among free-living primates. The viruses in the areas studied might have been introduced by infected humans or by primates from endemic or enzootic areas. Studies of this nature, as well as efficient and continuous surveillance programs, are needed to monitor viral activities in endemic and enzootic regions.

An optimized one-tube, semi-nested PCR assay for Paracoccidioides brasiliensis detection

Introduction Herein, we report a one-tube, semi-nested-polymerase chain reaction (OTsn-PCR) assay for the detection of Paracoccidioides brasiliensis. Methods We developed the OTsn-PCR assay for the detection of P. brasiliensis in clinical specimens and compared it with other PCR methods. Results The OTsn-PCR assay was positive for all clinical samples, and the detection limit was better or equivalent to the other nested or semi-nested PCR methods for P. brasiliensis detection. Conclusions The OTsn-PCR assay described in this paper has a detection limit similar to other reactions for the molecular detection of P. brasiliensis, but this approach is faster and less prone to contamination than other conventional nested or semi-nested PCR assays.

Detection of antibodies against Leishmania infantum in cats (Felis catus) from the State of Pernambuco, Brazil

Introduction: Little information is available concerning infection by Leishmania infantum in cats. Therefore, the aim of this study was to perform a serological study in domestic cats. Methods: Serum samples (n=153) obtained from animals living in the Cities of Recife and Petrolina, State of Pernambuco, Brazil, were tested by ELISA/S7® (Biogene). Results: Anti-L. infantum antibodies were detected in 3.9% (6/153) of the cats. All seroreagent animals were from Petrolina. Conclusions: These results serve as an important alert, and future studies are needed to better understand the possible role of cats in the epidemiology of visceral leishmaniasis (VL) in this area.

Hybrid capture II and PapilloCheck® tests for detection of anal high-risk human papillomavirus

Introduction This study evaluated the level of concordance between hybrid capture II (HCII) and PapilloCheck® for the detection of high-risk human papillomavirus (HPV) in anal samples. Methods Anal cell samples collected from 42 human immunodeficiency virus (HIV)+ patients were analyzed. Results Considering only the 13 high-risk HPV types that are detectable by both tests, HCII was positive for 52.3% of the samples, and PapilloCheck® was positive for 52.3%. The level of concordance was 80.9% (Kappa = 0.61). Conclusions Good concordance was observed between the tests for the detection of high-risk HPV.

A complete molecular biology assay for hepatitis C virus detection, quantification and genotyping

Introduction Molecular biology procedures to detect, genotype and quantify hepatitis C virus (HCV) RNA in clinical samples have been extensively described. Routine commercial methods for each specific purpose (detection, quantification and genotyping) are also available, all of which are typically based on polymerase chain reaction (PCR) targeting the HCV 5′ untranslated region (5′UTR). This study was performed to develop and validate a complete serial laboratory assay that combines real-time nested reverse transcription-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) techniques for the complete molecular analysis of HCV (detection, genotyping and viral load) in clinical samples. Methods Published HCV sequences were compared to select specific primers, probe and restriction enzyme sites. An original real-time nested RT-PCR-RFLP assay was then developed and validated to detect, genotype and quantify HCV in plasma samples. Results The real-time nested RT-PCR data were linear and reproducible for HCV analysis in clinical samples. High correlations (> 0.97) were observed between samples with different viral loads and the corresponding read cycle (Ct - Cycle threshold), and this part of the assay had a wide dynamic range of analysis. Additionally, HCV genotypes 1, 2 and 3 were successfully distinguished using the RFLP method. Conclusions A complete serial molecular assay was developed and validated for HCV detection, quantification and genotyping.

Detection of purkinje images for automatic positioning of fixation target and interferometric measurements of anterior eye chamber

In cataract surgery, the eye’s natural lens is removed because it has gone opaque and doesn’t allow clear vision any longer. To maintain the eye’s optical power, a new artificial lens must be inserted. Called Intraocular Lens (IOL), it needs to be modelled in order to have the correct refractive power to substitute the natural lens. Calculating the refractive power of this substitution lens requires precise anterior eye chamber measurements. An interferometry equipment, the AC Master from Zeiss Meditec, AG, was in use for half a year to perform these measurements. A Low Coherence Interferometry (LCI) measurement beam is aligned with the eye’s optical axis, for precise measurements of anterior eye chamber distances. The eye follows a fixation target in order to make the visual axis align with the optical axis. Performance problems occurred, however, at this step. Therefore, there was a necessity to develop a new procedure that ensures better alignment between the eye’s visual and optical axes, allowing a more user friendly and versatile procedure, and eventually automatizing the whole process. With this instrument, the alignment between the eye’s optical and visual axes is detected when Purkinje reflections I and III are overlapped, as the eye follows a fixation target. In this project, image analysis is used to detect these Purkinje reflections’ positions, eventually automatically detecting when they overlap. Automatic detection of the third Purkinje reflection of an eye following a fixation target is possible with some restrictions. Each pair of detected third Purkinje reflections is used in automatically calculating an acceptable starting position for the fixation target, required for precise measurements of anterior eye chamber distances.

Streptococcal acute pharyngitis

Acute pharyngitis/tonsillitis, which is characterized by inflammation of the posterior pharynx and tonsils, is a common disease. Several viruses and bacteria can cause acute pharyngitis; however, Streptococcus pyogenes (also known as Lancefield group A β-hemolytic streptococci) is the only agent that requires an etiologic diagnosis and specific treatment. S. pyogenes is of major clinical importance because it can trigger post-infection systemic complications, acute rheumatic fever, and post-streptococcal glomerulonephritis. Symptom onset in streptococcal infection is usually abrupt and includes intense sore throat, fever, chills, malaise, headache, tender enlarged anterior cervical lymph nodes, and pharyngeal or tonsillar exudate. Cough, coryza, conjunctivitis, and diarrhea are uncommon, and their presence suggests a viral cause. A diagnosis of pharyngitis is supported by the patient's history and by the physical examination. Throat culture is the gold standard for diagnosing streptococcus pharyngitis. However, it has been underused in public health services because of its low availability and because of the 1- to 2-day delay in obtaining results. Rapid antigen detection tests have been used to detect S. pyogenes directly from throat swabs within minutes. Clinical scoring systems have been developed to predict the risk of S. pyogenes infection. The most commonly used scoring system is the modified Centor score. Acute S. pyogenes pharyngitis is often a self-limiting disease. Penicillins are the first-choice treatment. For patients with penicillin allergy, cephalosporins can be an acceptable alternative, although primary hypersensitivity to cephalosporins can occur. Another drug option is the macrolides. Future perspectives to prevent streptococcal pharyngitis and post-infection systemic complications include the development of an anti-Streptococcus pyogenes vaccine.

Leprosy and pregnancy: detection coefficient and proposal for a new index

Introduction Our study presents a method to generate a novel detection coefficient for the association between leprosy and pregnancy (DCLP). Methods The DCLP was calculated for women from the State of Pará (2007-2009), Brazil. Data were ordered, divided into five equal parts (corresponding to the P20, P40, P60, and P80 percentiles), and classified as low, medium, high, very high, or hyperendemic. Results Using the new index, we established the DCLP parameters for low (<0.36), medium (0.36-0.69), high (0.70-1.09), very high (1.10-1.50), and hyperendemic (>1.50). Conclusions The new DCLP is more appropriate than the overall detection coefficient (DC), which does not take into account the particularities of the interaction between a disease and a specific physiological state.

Detection of intestinal parasites on field-grown strawberries in the Federal District of Brazil

Introduction This study evaluated the presence of pathogenic human parasites on field-grown strawberries in the Federal District of Brazil. Methods A total of 48 samples of strawberries and 48 soil samples from 16 properties were analyzed. Results Contaminated strawberries were detected in 56% of the properties. Schistosoma mansoni, Ascaris lumbricoides or Ascaris suum, Balantidium coli, Endolimax nana, and Entamoeba spp. were detected. Soil was contaminated with Entamoeba spp., Entamoeba coli, Strongyloides spp., Ancylostomatidae, and Hymenolepis nana. Conclusions Producers should be instructed on the safe handling of strawberries in order to reduce the incidence of strawberries that are contaminated with enteroparasites.

Leishmania, Babesia and Ehrlichia in urban pet dogs: co-infection or cross-reaction in serological methods?

INTRODUCTION: The present study was designed to assess the occurrence of co-infection or cross-reaction in the serological techniques used for detecting the anti-Leishmania spp., -Babesia canis vogeli and -Ehrlichia canis antibodies in urban dogs from an area endemic to these parasites. METHODS: The serum samples from dogs were tested for the Babesia canis vogeli strain Belo Horizonte antigen and Ehrlichia canis strain São Paulo by immunofluorescence antibody test (IFAT) and by anti-Leishmania immunoglobulin G (IgG) antibody detection to assess Leishmania infection. We used the following four commercial kits for canine visceral leishmaniasis: ELISA, IFAT, Dual Path Platform (DPP) (Bio Manguinhos(r)/FIOCRUZ/MS) and a rK39 RDT (Kalazar Detect Canine Rapid Test; Inbios). RESULTS : Of 96 serum samples submitted to serological assays, 4 (4.2%) were positive for Leishmania as determined by ELISA; 12 (12.5%), by IFAT; 14 (14.6%) by rK39 RDT; and 20 (20.8%), by DPP. Antibodies against Ehrlichia and Babesia were detected in 23/96 (23.9%) and 30/96 (31.2%) samples, respectively. No significant association was identified between the results of tests for detecting Babesia or Ehrlichia and those for detecting Leishmania (p-value>0.05). CONCLUSIONS: In the present study, we demonstrated co-infection with Ehrlichia or Babesia and Leishmania in dogs from Minas Gerais (Brazil); we also found that the serological tests that were used did not cross-react.

Detection of bla KPC-2 in Proteus mirabilis in Brazil

INTRODUCTION : Infections caused by Klebsiella pneumoniae carbapenemase (KPC)-producing isolates pose a major worldwide public health problem today. METHODS : A carbapenem-resistant Proteus mirabilis clinical isolate was investigated for plasmid profiles and the occurrence of β-lactamase genes. RESULTS : The isolate exhibited resistance to ertapenem and imipenem and was susceptible to meropenem, polymyxin, and tigecycline. Five plasmids were identified in this isolate. DNA sequencing analysis revealed the presence of bla KPC-2 and bla TEM-1 genes. An additional PCR using plasmid DNA confirmed that bla KPC-2 was present in one of these plasmids. Conclusions: We report the detection of bla KPC-2 in P. mirabilis in Brazil for the first time. This finding highlights the continuous transfer of bla KPC between bacterial genera, which presents a serious challenge to the prevention of infection by multidrug-resistant bacteria.

The use of saliva as a practical and feasible alternative to urine in large-scale screening for congenital cytomegalovirus infection increasesinclusion and detection rates

INTRODUCTION: Although urine is considered the gold-standard material for the detection of congenital cytomegalovirus (CMV) infection, it can be difficult to obtain in newborns. The aim of this study was to compare the efficiency of detection of congenital CMV infection in saliva and urine samples. METHODS: One thousand newborns were included in the study. Congenital cytomegalovirus deoxyribonucleic acid (DNA) was detected by polymerase chain reaction (PCR). RESULTS: Saliva samples were obtained from all the newborns, whereas urine collection was successful in only 333 cases. There was no statistically significant difference between the use of saliva alone or saliva and urine collected simultaneously for the detection of CMV infection. CONCLUSIONS: Saliva samples can be used in large-scale neonatal screening for CMV infection.

Detection of immunogenic proteins from Anopheles sundaicussalivary glands in the human serum

AbstractINTRODUCTION:The saliva of mosquitoes has an important role in the transmission of several diseases, including malaria, and contains substances with vasomodulating and immunomodulating effects to counteract the host physiological mechanisms and enhance pathogen transmission. As immunomodulatory components, salivary gland proteins can induce the generation of specific IgG antibodies in the host, which can be used as specific biomarkers of exposure to Anopheles sundaicus . The objective of this study was to identify immunogenic proteins from the salivary glands of Anopheles sundaicus by reaction with sera from individuals living in malaria-endemic areas who are thus exposed to Anopheles mosquitoes.METHODS:IgG antibodies targeting salivary gland proteins in serum samples from individuals living in malaria-endemic areas were measured by enzyme-linked immunosorbent assay (ELISA). Sera from healthy individuals living in non-endemic areas were used as negative controls. Determination of the presence of salivary gland immunogenic proteins was carried out by western blotting.RESULTS:Sixteen bands appeared in sodium dodecyl sulfate polyacrylamide gel electrophoresis, with molecule weights ranging from 22 to 144kDa. Among the exposed individuals, IgG responses to salivary gland proteins were variable. Protein bands with molecular weights of 46, 41, 33, and 31kDa were the most immunogenic. These immunogenic proteins were consistently recognized by pooled serum and individual samples from people living in malaria-endemic areas but not by negative controls.CONCLUSIONS:These results support the potential use of immunogenic proteins from the salivary glands of Anopheles as candidate markers of bite exposure or in malaria vaccines.