Comparison of nested polymerase chain reaction and real-time polymerase chain reaction targeting 47kda gene for the diagnosis of scrub typhus

Introduction: Scrub typhus is a zoonotic infection caused by Orientia tsutsugamushi which is transmitted by Leptotrombidium mites. The disease manifests as a mild-to-severe illness with non-specific clinical symptoms. Rapid diagnosis and prompt treatment are essential for patient management. Both serological and molecular methods are used for the diagnosis of scrub typhus. The present study assessed the usefulness of detection of the gene encoding the 47kDa outer-membrane protein (OMP) for the laboratory diagnosis of scrub typhus. Materials and Methods: Nested polymerase chain reaction (nPCR) and real-time PCR targeting 47 kDa OMP antigen gene of O. tsutsugamushi were performed on ethylenediaminetetraacetic acid blood samples. Results: Six of the 103 (5.8%) patients showed the presence of 47kDa gene by nPCR. Seventy of 103 (67.9%) cases showed the presence of 47kDa gene by qPCR. Among the 70 positive cases, the majority of them were females (40/70, 57.1%). The highest number of positive cases was observed during October朏ebruary. Conclusion: Real-time PCR targeting O. tsutsugamushi-specific 47-kDa gene is more sensitive than nPCR and may be the assay of choice for the detection of the organism in patients with suspected scrub typhus.

Detection of parvovirus B19 in selected high-risk patient groups & their phylogenetic & selection analysis

Background & objectives: Human parvovirus B19V (B19V) is known to be associated with erythema infectiosum commonly in children, aplastic crisis, especially in persons with underlying haemolytic disorders, hydrops fetalis in pregnancies and arthritis. This cross-sectional study was aimed to determine the presence of B19V infection in childhood febrile illnesses, association of B19V with arthropathies and in adult patients with end-stage renal disease (ESRD) on dialysis. The genetic diversity among the sequences was also analysed. Methods: A nested polymerase chain reaction (nPCR) assay was used for B19V DNA targeting VP1/VP2 region and used for testing 618 patients and 100 healthy controls. Phylogenetic analysis on nucleotide and amino acid sequences was carried out to compare our sequences with other Indian strains and global strains. Results: Among 618 samples tested, seven (1.13%) were found positive. The phylogenetic analysis revealed that all the seven sequences belonged to genotype 1 and showed low genetic diversity. The clustering pattern of seven sequences was similar both by nucleotide and by predicted amino acid sequences. The fixed effects likelihood analysis showed no positive or negatively selected sites. Interpretation & conclusions: Seven samples (4 from non-traumatic arthropathies, 2 from patients with ESRD and 1 from febrile illness patient) were found positive by nPCR. When our seven sequences were compared with global strains, the closest neighbour was other Indian strains followed by the Tunisian strains.

Anaplasma phagocytophilum direct detection and exposure evidence in equines from two breeding farms from Minas Gerais State, Brazil / Detecção direta e evidência da exposição a Anaplasma phagocytophilum em equinos de dois haras em Minas Gerais, Brasil

Equine granulocytic anaplasmosis is caused by Anaplasma phagocytophilum, a gram negative, obligatory intracellular bacterium, member of Anaplasmataceae family, included in the Rickettsiales order. Little is known about the disease, transmission dynamics, genetic diversity and prevalence in Minas Gerais state, Brazil. This work aimed to do a serosurvey using indirect immunofluorescent assay (IFA) test and evaluation of buffy coat smears, and nested polymerase chain reaction (PCR) as diagnostic methods, to determine the disease situation in horses from two manga-larga marchador breeding farms located in the municipalities of Ataléia e São Vicente de Minas, in Minas Gerais state. It was found that 76% (131/172) of the animals were considered reactive for IFA test, and the total of 12.8% was positive at buffy coat smears analysis. At PCR analysis, it was found 1.94% of the samples positive to the infection. Those samples were sequenced and showed 96% of similarity to A. phagocytophilum from a Ixodes ricinus tick. There is a high frequency of animals with the evidence of contact to A. phagocytophilum on the two evaluated properties in this study, which was proved by positiveness in PCR analysis. New researches must be carried out to better understand the epidemiologic and clinical dynamic of the disease in the state of Minas Gerais.(AU)

A anaplasmose granulocítica equina é causada por uma bactéria gram-negativa, intracelular obrigatória, membro da família Anaplasmataceae, incluída na ordem Rickettsiales e denominada de Anaplasma phagocytophilum. Pouco se sabe sobre a doença, sua dinâmica de transmissão, diversidade genética e prevalência em Minas Gerais, Brasil. Este trabalho teve por objetivo realizar o levantamento sorológico utilizando a reação de imunofluorescência indireta, avaliação direta de capa leucocitária e nested reação em cadeia da polimerase (PCR) como métodos diagnósticos, a fim de avaliar a situação da doença em dois haras de criação de cavalos manga-larga marchador localizados nas cidades de Ataléia e São Vicente de Minas, no estado de Minas Gerais. Foi encontrada prevalência de 76% (131/172) de animais reativos para a reação de imunofluorescência indireta, quando todos os animais das duas propriedades e das duas coletas foram agrupados, e 12,8% dos animais foram positivos na avaliação da capa leucocitária. A reação de imunofluorescência indireta detectou 1,94% das amostras como positivas para o agente. Essas amostras foram submetidas ao sequenciamento de nucleotídeos, e foi observada similaridade de 96% com A. phagocytophilum proveniente de carrapatos Ixodes ricinus. Existe alta prevalência de animais positivos para a infecção por A. phagocytophilum, o que foi provado pela positividade dos animais à PCR. Novas pesquisas devem ser conduzidas a fim de entender a dinâmica epidemiológica e clínica da doença no estado de Minas Gerais.(AU)

Application of nested real-time PCR in detecting Treponema palladium DNA in various clinical samples from patients preliminarily diagnosed as syphilis / 中华皮肤科杂志

Objective To investigate the feasibility and prospects of nested real-time PCR(NR-PCR)technique for Treponema palladium(Tp)detection in various samples of different stages of syphilis from patients preliminarily diagnosed as syphilis. Methods Targeting the Tp polA gene, NR-PCR was performed to detect Tp DNA in various samples from the patients with various stages of syphilis at the first clinic visit, including skin tissue fluid swabs, serum, whole blood, cerebrospinal fluid(CSF)and earlobe blood. Data were analyzed with SPSS software version 13. Results A total of 368 clinical samples were collected from 200 patients with syphilis. With a detection limit of 2 Tp/ml, NR-PCR showed that the total positive rate for Tp DNA was 71.7%(264/368). The Tp DNA positive rate was highest in earlobe blood samples (92.0%, 23/25), followed by CSF samples(90.2%, 46/51), skin tissue fluid swabs(74.3%, 26/35), serum samples(66.9%, 99/148)and whole blood samples(64.2%, 70/109). There was good agreement between NR-PCR results and serologic test results, with a consistency rate of 76.0%(152/200). Furthermore, the Tp DNA positive rate did not differ between patients with primary(12/19)and secondary syphilis(14/16)in skin tissue fluid swabs(χ2 = 2.62, P > 0.05), and was slightly but insignificantly higher in patients with secondary syphilis than those with primary syphilis in the serum samples(χ2=3.6, P=0.06). The Tp DNA positive rate of whole blood samples was also higher in patients with secondary syphilis than those with any other types of syphilis. Among patients with neurosyphilis, no significant difference was observed in the Tp DNA positive rate between earlobe blood samples and CSF samples(P=0.06). Among patients with latent syphilis, the Tp DNA positive rate was significantly higher in serum samples with an RPR titer of ≥ 1:8 than those with an RPR titer of≤1:4. Conclusion NR-PCR is feasible for detecting Tp DNA in various kinds of samples, and the Tp DNA positive rate is influenced by stages of syphilis and types of samples, as well as RPR titers.

Analysis on etiological diagnosis of first case of imported Plasmodium ovale wallikeri subspecies in Hubei Province / 国际检验医学杂志

Objective To use the nested PCR technology to diagnose and identify a case of imported Plasmodium ovale wallikeri subspecies .Methods Blood sample was collected from 1 case of initially diagnosed imported tertian malaria and performed the ex‐amination of microscopy ,rapid diagnostic tests (RDTs) and nested‐PCR .Moreover the sequencing was conduced .Results RDTs showed the negative result ;the ring form and trophozoite of Plasmodium could be observed in the blood smear by microscopy ;the Plasmodium ovale wallikeri subspecies specific primer rOVA 1v/rOVA2v was adopted for conducting nested PCR ,the specicific am‐plification band appeared at 760 bp ,after sequencing and Blast aligning ,its coincidence with the partial sequence of Plasmodium ovale wallikeri subspecies in the Genbank database was 99% .Conclusion This patient is the first case of Plasmodium ovale wallik‐eri subspecies infection in Hubei province by nested PCR and sequencing analysis .

Single-tube nested PCR assay with in-house DNA extraction for Mycobacterium tuberculosis detection in blood and urine

Abstract: INTRODUCTION : Molecular analyses are auxiliary tools for detecting Koch's bacilli in clinical specimens from patients with suspected tuberculosis (TB). However, there are still no efficient diagnostic tests that combine high sensitivity and specificity and yield rapid results in the detection of TB. This study evaluated single-tube nested polymerase chain reaction (STNPCR) as a molecular diagnostic test with low risk of cross contamination for detecting Mycobacterium tuberculosis in clinical samples. METHODS: Mycobacterium tuberculosis deoxyribonucleic acid (DNA) was detected in blood and urine samples by STNPCR followed by agarose gel electrophoresis. In this system, reaction tubes were not opened between the two stages of PCR (simple and nested). RESULTS: STNPCR demonstrated good accuracy in clinical samples with no cross contamination between microtubes. Sensitivity in blood and urine, analyzed in parallel, was 35%-62% for pulmonary and 41%-72% for extrapulmonary TB. The specificity of STNPCR was 100% in most analyses, depending on the type of clinical sample (blood or urine) and clinical form of disease (pulmonary or extrapulmonary). CONCLUSIONS: STNPCR was effective in detecting TB, especially the extrapulmonary form for which sensitivity was higher, and had the advantage of less invasive sample collection from patients for whom a spontaneous sputum sample was unavailable. With low risk of cross contamination, the STNPCR can be used as an adjunct to conventional methods for diagnosing TB.

Occurrence of Cryptosporidium spp. and Giardia spp. in a public water-treatment system, Paraná, Southern Brazil / Ocorrência de Cryptosporidium spp. e Giardia spp. em uma estação pública de tratamento de água, Paraná, Sul do Brasil

The purpose of this study was to investigate the occurrence of Cryptosporidium spp. and Giardia spp. in a public water-treatment system. Samples of raw and treated water were collected and concentrated using the membrane filtration technique. Direct Immunofluorescence Test was performed on the samples. DNA extraction using a commercial kit was performed and the DNA extracted was submitted to a nested-PCR reaction (n-PCR) and sequencing. In the immunofluorescence, 2/24 (8.33%) samples of raw water were positive for Giardia spp.. In n-PCR and sequencing, 2/24 (8.33%) samples of raw water were positive for Giardia spp., and 2/24 (8.33%) samples were positive for Cryptosporidium spp.. The sequencing showed Cryptosporidium parvum and Giardia duodenalis DNA. In raw water, there was moderate correlation among turbidity, color and Cryptosporidium spp. and between turbidity and Giardia spp.. The presence of these protozoans in the water indicates the need for monitoring for water-treatment companies.

O objetivo deste estudo foi investigar a ocorrência de Cryptosporidium spp. e Giardia spp. em um sistema público de tratamento de água. Amostras de água bruta e tratada foram coletadas e concentradas, utilizando-se a técnica de filtração em membranas. Foi realizada a técnica de Imunofluorescência Direta nas amostras. A extração de DNA foi realizada, utilizando-se um kit comercial, e o DNA extraído foi submetido a uma reação de nested-PCR (n-PCR). Na imunofluorescência, 2/24 (8,33%) amostras de água bruta foram positivas para Giardiaspp.. Na n-PCR, 2/24 (8,33%) amostras de água bruta foram positivas para Giardia spp., e 2/24 (8,33%) amostras foram positivas para Cryptosporidium spp.. O sequenciamento demonstrou DNA de Cryptosporidium parvum e de Giardia duodenalis. Na água bruta, houve correlação moderada entre turbidez, cor e Cryptosporidium spp. e entre a turbidez e Giardia spp.. A presença desses protozoários na água indica a necessidade de monitoramento pelas empresas de tratamento de água.

An epidemiological comparative study on diagnosis of rodent leptospirosis in Mazandaran Province, northern Iran / 한국역학회지

OBJECTIVES: Leptospirosis is a zoonosis caused by leptospires, in which transmission occurs through contact with contaminated biological fluids from infected animals. Rodents can act as a source of infection for humans and animals. The disease has a global distribution, mainly in humid, tropical and sub-tropical regions. The aim of this study was to compare culture assays, the microscopic agglutination test (MAT), polymerase chain reaction (PCR), and nested PCR (n-PCR), for the diagnosis of leptospirosis in rodents in Mazandaran Province, northern Iran. METHODS: One hundred fifty-one rodents were trapped alive at 10 locations, and their urine and kidney samples were collected and used for the isolation of live Leptospira. The infecting serovars were identified and the antibody titres were measured by MAT, using a panel of 20 strains of live Leptospira species as antigens. The presence of leptospiral DNA was evaluated in urine and kidney samples using PCR and n-PCR. RESULTS: No live leptospires were isolated from the kidney and urine samples of the rodents. Different detection rates of leptospirosis were observed with MAT (21.2%), PCR (11.3%), and n-PCR (3.3%). The dominant strain was Leptospira serjoehardjo (34.4%, p=0.28), although other serotypes were also found. The prevalence of positive leptospirosis tests in rodents was 15.9, 2.6, and 2.6% among Rattus norvegicus, R. rattus, and Apodemus sylvaticus, respectively. CONCLUSIONS: Leptospirosis was prevalent in rodents in Mazandaran Province, northern Iran. MAT was able to detect leptospires more frequently than culture or PCR. The kidney was a more suitable site for identifying leptospiral DNA by n-PCR than urine. Culture was not found to be an appropriate technique for clinical diagnosis.

In vitro interaction of bovine herpesvirus 1 with uterine tube epithelial cells and oocytes / Interação in vitro do herpesvírus bovino tipo 1 com células epiteliais da tuba uterina e oócitos

The aims of this study were to assess in vitro if bovine oocytes and oviductal epithelial cells from slaughterhouses for in vitro fertilization use may be infected with bovine herpesvirus 1; to analyze whether the treatment with trypsin according to the International Embryo Transfer Society guideline is efficient to inactivate the bovine herpesvirus 1; to morphologically study the virus-oocyte interaction through optical microscopy. In this study, Madin Darby Bovine Kidney (MDBK) cells that were co-cultured with oocytes matured in vitro and exposed to bovine herpesvirus 1 showed a cytopathic effect. The nested polymerase chain reaction for the supernatant was positive for the bovine herpesvirus 1, thus suggesting that the cytopathic effect observed in the MDBK monolayer was seen due to virus replication and not because of any culture toxicity. It was also observed cytopathic effect and positive nested polymerase chain reaction in MDBK cells co-cultured with in vitro maturated oocytes free of virus, but that were co-cultured in uterine epithelial cells pre-infected with bovine herpesvirus 1 and washed or not with trypsin, demonstrating an oocyte contamination by the virus. When trypsin-washing efficacy was evaluated, we could observe that the trypsin treatment was not able to eliminate the bovine herpesvirus 1 of the oocytes, and it was not observed any morphological difference in the infected oocytes.(AU)

Os objetivos do presente estudo foram avaliar in vitro se oócitos bovinos e células epiteliais de oviduto provenientes de abatedouros para uso em fertilização in vitro podem ser infectados com o herpesvírus bovino tipo 1; analisar se o tratamento com tripsina padronizado pelo International Embryo Transfer Society é eficiente para inativar o herpesvírus bovino tipo 1; estudar morfologicamente a interação vírus e oócito pela microscopia óptica. Neste estudo, as células Madin Darby Bovine Kidney (MDBK), que foram cocultivadas com oócitos maturados in vitro e expostos ao herpesvírus bovino tipo 1, apresentaram efeito citopático. A reação em cadeia da polimerase aninhada ao sobrenadante foi positiva para o herpesvírus bovino tipo 1, sugerindo que o efeito citopático observado na monocamada MDBK foi em função da replicação do vírus, mas não devido a qualquer toxicidade da cultura. Também foram mostrados efeito citopático e reação em cadeia da polimerase aninhada positivos em células MDBK cocultivadas com oócitos maturados in vitro isentos de vírus, porém que foram cocultivados em células epiteliais uterinas previamente infectadas com herpesvírus bovino tipo 1, que se lavou ou não com tripsina, demonstrando uma contaminação pelo vírus do oócito. Quando foi avaliada a eficácia de lavagem com a tripsina, foi possível notar que este tratamento não foi capaz de eliminar o herpesvírus bovino tipo 1 dos oócitos, e não foi observada qualquer diferença morfológica nos oócitos infectados.(AU)

Diagnóstico de neumocistosis a través de la inmunofluorescencia directa y la PCR anidada en pacientes con enfermedad pulmonar obstructiva crónica / Diagnosis through pneumocystosis direct immunofluorescence and nested PCR in patients with chronic obstructive pulmonary disease

La asociación EPOC- neumocitosis está descrita y existe la necesidad de optimizar la diferenciación entre enfermedad y colonización. Demostrar la presencia del Pneumocistys Jirovecci, como patógeno y/o colonizador. Estudio descriptivo, analítico, de cohorte de pacientes con diagnóstico de EPOC del Hospital General del Oeste (Caracas, Venezuela) durante el periodo de abril – julio 2012 con seguimiento hasta julio de 2013 y aplicación de la técnica de inmunofluorescencia directa (IFD) y/o PCR anidada (PCRa) en muestra de esputo (espontáneo – inducido) durante los periodos asintomáticos o durante la exacerbación del EPOC en seguimiento de un año. Se incluyeron 20 pacientes en el reclutamiento, con seguimiento al primer control de 5 pacientes; de estos solo 2 cumplieron la medición de esputo. Para la tercera evaluación una paciente había fallecido y la otra no cumplió con el seguimiento. Se demostró IFI+ en 10% de los reclutados, todos con clínica de exacerbación de la EPOC. La PCRa se demostró en 45%, 2 con exacerbación y el resto sin exacerbación. De los dos pacientes de seguimiento, una fue positiva para PCRa y no tenía exacerbación, la otra negativa por ambos métodos. Se demostró infección por Pj en los pacientes con EPOC exacerbado a través de IFI y la PCRa señala su positividad en infección pero también en aquellos sin infección o exacerbación documentando así la colonización y potencial fuente de infección para neumocistosis. Se demostró infección por Pj en paciente con exacerbación y colonización a través de la evidencia del genoma del hongo en pacientes sin exacerbación.

Pneumocistosis and COPD association is described and there is a need to differenciate between disease and colonization. To document the presence of Pnemocistys jirovecci as pathogenic or colonizer by direct immunofluoresencence technique (DIF) and/or nested polymerase chain reaction (nPCR) in sputum (spontaneous-induced) during asymptomatic periods or exacerbation of COPD during a year of follow-up. This is a a descriptive, analytic cohort of patients with COPD of the Hospital General del Oeste (Caracas, Venezuela) . They were studied during April - July 2012, with follow-up until July 2013. 20 patients were included. The first control follow up was in 5 patients with only two measures of IFI - PCRa. For the third evaluation one patient had died and the other did not comply with control. IFI + was demonstrated in 10 % of the recruits, all had COPD, exacerbation. PCRa + was demonstrated in 45%, 2 with exacerbation and all other without exacerbation. From the two followed patients one was positive for PCRn and had no exacerbation, the other was negative by both methods. Pj infection was demonstrated in patients with exacerbated COPD by IFI+ and the PCRa positivity in infection but also in those without infection or exacerbation documenting the colonization and potential source of infection for Pj. Pj infection wasdiagnosed in patients with exacerbation COPD and colonization through the evidence of the genome of the fungus in patients without exacerbation.

Clinical, radiological and molecular diagnosis correlation in serum samples from patients with osteoarticular tuberculosis

<p><b>OBJECTIVE</b>To assess the role of polymerase chain reaction (PCR) in serum samples, in the diagnosis of osteoarticular tuberculosis (OTB) in a setting where only clinical and imaging diagnoses determine the treatment.</p><p><b>METHODS</b>A total of 44 consecutive serum specimens were collected from clinically suspected OTB patients, based on clinical and radiological [X-ray or magnetic resonance imaging/computed tomography] features. They were screened by in-house nested PCR. In addition, a few specimens were examined by Gram stain, acid-fast bacilli stain, histopathology and routine bacterial culture. A total of 39 specimens were collected from patients suffering from other bone diseases of nontuberculous origin and included as negative controls.</p><p><b>RESULTS</b>Of the 44 clinically suspected OTB patients, in-house nested PCR was positive in 40 (91%) cases; PCR was negative in 38 (97%) negative controls. Sensitivity and specificity of our in-house nested PCR was 90.9% and 97.4%, respectively. The PCR report was available within 48 h. It was possible to standardize serum PCR technique and in positive cases, a good correlation was observed in terms of an adequate treatment response.</p><p><b>CONCLUSIONS</b>Nested PCR in serum samples is a rapid, highly sensitive and specific modality for OTB detection. PCR should be performed in addition to clinical evaluation, imaging studies, acid-fast bacilli staining, culture and histopathology diagnosis, if possible.</p>

Clinical, radiological and molecular diagnosis correlation in serum samples from patients with osteoarticular tuberculosis

Objective: To assess the role of polymerase chain reaction (PCR) in serum samples, in the diagnosis of osteoarticular tuberculosis (OTB) in a setting where only clinical and imaging diagnoses determine the treatment.Methods:A total of 44 consecutive serum specimens were collected from clinically suspected OTB patients, based on clinical and radiological [X-ray or magnetic resonance imaging/computed tomography] features. They were screened by in-house nested PCR. In addition, a few specimens were examined by Gram stain, acid-fast bacilli stain, histopathology and routine bacterial culture. A total of 39 specimens were collected from patients suffering from other bone diseases of nontuberculous origin and included as negative controls.Results:Of the 44 clinically suspected OTB patients, in-house nested PCR was positive in 40 (91%) cases; PCR was negative in 38 (97%) negative controls. Sensitivity and specificity of our in-house nested PCR was 90.9% and 97.4%, respectively. The PCR report was available within 48 h. It was possible to standardize serum PCR technique and in positive cases, a good correlation was observed in terms of an adequate treatment response.Conclusions:Nested PCR in serum samples is a rapid, highly sensitive and specific modality for OTB detection. PCR should be performed in addition to clinical evaluation, imaging studies, acid-fast bacilli staining, culture and histopathology diagnosis, if possible.

Clinical, radiological and molecular diagnosis correlation in serum samples from patients with osteoarticular tuberculosis

Objective: To assess the role of polymerase chain reaction (PCR) in serum samples, in the diagnosis of osteoarticular tuberculosis (OTB) in a setting where only clinical and imaging diagnoses determine the treatment. Methods: A total of 44 consecutive serum specimens were collected from clinically suspected OTB patients, based on clinical and radiological [X-ray or magnetic resonance imaging/computed tomography] features. They were screened by in-house nested PCR. In addition, a few specimens were examined by Gram stain, acid-fast bacilli stain, histopathology and routine bacterial culture. A total of 39 specimens were collected from patients suffering from other bone diseases of nontuberculous origin and included as negative controls. Results: Of the 44 clinically suspected OTB patients, in-house nested PCR was positive in 40 (91%) cases; PCR was negative in 38 (97%) negative controls. Sensitivity and specificity of our in-house nested PCR was 90.9% and 97.4%, respectively. The PCR report was available within 48 h. It was possible to standardize serum PCR technique and in positive cases, a good correlation was observed in terms of an adequate treatment response. Conclusions: Nested PCR in serum samples is a rapid, highly sensitive and specific modality for OTB detection. PCR should be performed in addition to clinical evaluation, imaging studies, acid-fast bacilli staining, culture and histopathology diagnosis, if possible.

Difference between Nested-polymerase chain reaction and virus isolation in detection of respiratory syncytial virus and their clinical significances / 中华实用儿科临床杂志

Objective To observe the differences between Nested-polymerase chain reaction(N-PCR) and virus isolation methods used for detection of respiratory syncytial virus(RSV),and to reveal the potential clinical features of them.Methods From Jan.2010 to Aug.2012,nasopharyngeal aspirates (NPAs) were collected from the children with respiratory infection in the Department of Respiratory,the Children's Hospital of Chongqing Medical University.Both N-PCR and virus isolation were applied to detect RSV,and clinical data were collected for statistical analysis.Results A total of 1143 specimens were used for RSV detection by N-PCR and virus isolation.The male-female ratio was 2.16 vs 1.00.The age of patients was ranged from 1 month to 165 months(median:7 months).The most common diagnoses were as follows:bronchopneumonia [478 cases (41.8％)],chronic fibrous pneumonia [223 cases (19.5％)],bron-chiolitis [221 cases (19.3％)],bronchitis [71 cases (6.2％)] and upper respiratory infection [21 cases(1.8％)].For N-PCR,458 cases were RSV positive (total positive rate was 40.1％ ; 31.7％ for RSV-A,7.7％ for RSV-B,0.7％ for both RSV-A and RSV-B).With virus isolation method,204 cases were positive (17.8％).Comparison result of N-PCR and virus isolation showed:165 cases were positive (P+ I+) and 646 cases were negative (P-I-) by both methods (identity was 70.1％),and the most difference was N-PCR positive but virus isolation negative group (P+ I-) (293 cases,25.6％).When compared to P-I-group,the clinical features of P+ I-group were as follows:younger,longer hospital stays,remarkable season distribution (with peak in winter and lowest in summer),lower percentage of fever,higher percentage of cough,wheezing,dyspnea,severe pneumonia and respiratory failure,all these differences were statistically significant(all P ＜ 0.05),the ma-nifestations matched the clinical features of RSV infection.When compared to P + I + group,the symptoms in the P + I-group had longer duration before they were admitted to hospital (P =0.005) and lower percentage of wheezing (P =0.009).Conclusions The differences between N-PCR and virus isolation for the detection of RSV existed in duration of symptoms prior to hospitalization.Both the sensibility and specificity of N-PCR are desirable for RSV detection.

Standardization of fungal polymerase chain reaction for the early diagnosis of invasive fungal infection.

Background: An early initiation of antifungal therapy in invasive fungal infections (IFIs) is critical in reducing the high mortality rate. Current diagnosis of fungal infection relies on microscopy, culture, antigen, antibody specific tests and histological diagnosis. However, these tests either lack sensitivity or specificity. There is thus the need for a rapid, specific and accurate diagnostic method. Objective: The aim of our study was to establish PCR for the rapid detection of Candida and Aspergillus species in clinical specimens with improved sensitivity and specificity. Materials and Methods: A total of 71 proven cases of IFI (confirmed by culture) were collected. A total of 15 healthy, 15 patients suffering from bacterial sepsis and 15 patients with HIV, HBV viral infections were included as controls. Clinical specimens were subjected to a standardized nested amplification to produce Round I (504 bp) and Round II (150 bp) amplicons. Restriction digestion was performed on these products for further identification. Results: Analytical sensitivity was determined using 10 6 -10 CFU/ml of cell suspension. The lower detection limit of the assay was 10 CFU/ml of blood. This test was 100% sensitive and specific with a positive predictive value of 100% and a negative predictive value of 96.7%. Conclusion: The assay was found to be effective for the rapid detection of Candida and Aspergillus in clinical specimens.

A study of tubercular lymphadenitis: A comparison of various laboratory diagnostic modalities with a special reference to tubercular polymerase chain reaction.

Objective: The purpose of our study was to compare various laboratory diagnostic methods, namely histopathological examination, Ziehl-Neelsen (ZN) stain, AFB culture by conventional Lowenstein-Jensen (LJ) method and fluorescence-based mycobacterial growth indicator tube (MGIT) technique and polymerase chain reaction (PCR) in clinically suspected cases of tubercular lymphadenitis. Materials and Methods: A total of 65 lymph nodes biopsied from patients clinically suspected of having tubercular lymph nodes were included. Specimens were processed for AFB culture after NaOH-NALC concentration and inoculation on LJ medium and using the MGIT system. PCR was performed on all specimens using a commercial nested PCR kit targeting IS6110 insertion element of Mycobacterium tuberculosis complex. All lymph node specimens were subjected to histopathological examination. Results: Of the 65 lymph nodes, 37 (56.9%) were positive on MGIT culture and 45 (69.2%) were positive by PCR. Histopathology showed maximum sensitivity (96%) but with compromised specificity (78.5%). PCR showed 90.1% sensitivity and 100% specificity. The mean turnaround time for mycobacterial growth in smear negative specimens was 30 days determined by LJ and 20 days by MGIT techniques. Conclusion: PCR is a rapid and useful method for diagnosis of TB lymphadenitis and definitely increases the positive predictive value of a positive histopathology report. MGIT is better than LJ culture as regards time to positivity and higher yield.

Nested polymerase chain reaction on blood clots for gene encoding 56 kDa antigen and serology for the diagnosis of scrub typhus.

Purpose : Scrub typhus is a zoonotic illness endemic in the Asia-Pacific region. Early diagnosis and appropriate management contribute significantly to preventing adverse outcomes including mortality. Serology is widely used for diagnosing scrub typhus. Recent reports suggest that polymerase chain reaction (PCR) could be a rapid and reliable alternative. This study assessed the utility of these tests for scrub typhus diagnosis. Materials and Methods : Nested PCR to detect the 56 kDa antigen gene of O. tsutsugamushi was performed on blood clots from 87 individuals with clinically suspected scrub typhus. Weil-Felix test and scrub typhus IgM ELISA were performed on serum samples from the same patients. As a gold standard reference test was not available, latent class analysis (LCA) was used to assess the performance of the three tests. Results : The LCA analysis showed the sensitivity of Weil-Felix test, IgM ELISA and PCR to be 59%, 100% and 58% respectively. The specificity of ELISA was only 73%, whereas those of the Weil-Felix test and PCR were 94% and 100% respectively. Conclusion : Nested PCR using blood clots while specific, lacked sensitivity as compared to IgM ELISA. In resource-poor settings Weil-Felix test still remains valuable despite its moderate sensitivity.

Detection of Varicella-Zoster Virus (VZV) by Nested Polymerase Chain Reaction / 임상검사와정도관리

BACKGROUND: Polymerase chain reaction (PCR) is known as a sensitive and specific method for the detection of varicella-zoster virus (VZV). Nested PCR is reliably used than conventional PCR to increase the sensitivity and specificity, especially in cases of small sized tissue samples. METHODS: We detected VZV infection in tissues from 111 patients using conventional PCR and nested PCR. Ninety-one cases of fresh tissues and twenty cases of formalin-fixed paraffin-embedded (FFPE) tissues were evaluated. The column method or home made lysis buffer method was used for the DNA extraction of fresh tissues and FFPE tissues. RESULTS: Among total 111 cases, VZV were detected in 62 (55.9%) cases by conventional PCR and 79 (71.2%) cases by nested PCR. The detection rate of nested PCR was higher than conventional PCR (1.27 folds). In 91 cases of fresh tissues, 56 (61.5%) were positive by conventional PCR and 68 (74.7%) by nested PCR. In 20 cases of FFPE tissues, 6 (30%) were positive by conventional PCR and 11 (55%) by nested PCR. The detection rate of VZV was increased by nested PCR both in fresh tissues (1.21 folds) and FFPE tissues (1.83 folds). CONCLUSIONS: Nested PCR is the more sensitive method than conventional PCR for the detection of VZV infection in tissues regardless of DNA extraction methods, especially for the small sized FFPE tissues.

Clinical usefulness of the nested polymerase chain reaction in the diagnosis of extrapulmonary tuberculosis / Utilidad clínica de la reacción en cadena de la polimerasa anidada para el diagnóstico de tuberculosis extrapulmonar

OBJECTIVE:To evaluate the effectiveness of nested polymerase chain reaction (PCR) for diagnosis of extrapulmonary tuberculosis (ETB), as well as the impact of PCR results on clinical management. MATERIALS AND METHODS:We conducted a study of nested PCR tests in 45 patients and a review of patient hospital files, calculating sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). RESULTS:PCR was positive in 51% of cases; PCR sensitivity for diagnosing TB was 86%, specificity was 79%, PPV was 76%, and NPV was 88%. When solely analyzing urine samples, sensitivity and NPV increased to 100%. PCR exerted an influence on management in 27% of patients. CONCLUSIONS:PCR for rapid diagnosis of extrapulmonary TB has an adequate effect, which improves when performed on urine. The results of PCR exerted an acceptable impact on the clinical management of these patients.

OBJETIVO:Evaluar la eficacia de la reacción en cadena de la polimerasa (PCR) anidada para el diagnóstico de tuberculosis extrapulmonar, así como el impacto de sus resultados en el manejo clínico. MATERIAL Y MÉTODOS: Se realizó PCR anidada en 45 pacientes y se llevó a cabo la revisión de expedientes. Se calculó sensibilidad, especificidad, valor predictivo positivo (VPP) y valor predictivo negativo (VPN). RESULTADOS:La PCR fue positiva en 51% de los casos, la sensibilidad fue de 86%, la especificidad de 79%, el VPP de 76% y el VPN de 88%. Al analizar solamente las muestras de orina, la sensibilidad y VPN se incrementaron a 100%. La PCR influyó en el manejo de 27% de los pacientes. CONCLUSIONES:La PCR para el diagnóstico rápido de TB extrapulmonar tiene una eficacia adecuada, la cual mejora cuando se realiza en orina. El resultado de la PCR tuvo un impacto aceptable en el manejo clínico de estos pacientes.

Detection of human herpesvirus-7 by qualitative nested-PCR: comparison between healthy individuals and liver transplant recipients / Detecção de herpesvirus humano-7 por nested-PCR qualitativo: comparação entre indivíduos sadios e receptores de transplante hepático

Diagnosis of human herpesvirus-7 active infection in transplant patients has proved difficult, because this virus is ubiquitous and can cause persistent infections in the host. The significance of viral DNA detected in leukocytes by PCR is unclear and cross-reaction in serological tests may occur. This study aimed to evaluate nested-PCR to detect human herpesvirus-7 active infection in liver transplant recipients compared to healthy individuals. human herpesvirus-7 nested-PCR was performed on leukocytes and sera of 53 healthy volunteers and sera of 29 liver transplant recipients. In healthy volunteers, human herpesvirus-7 was detected in 28.3 percent of leukocytes and 0 percent of serum. human herpesvirus-7 was detected in sera of 48.2 percent of the liver transplant recipients. Nested-PCR on DNA extracted from leukocytes detected latent infection and the study suggests that nested-PCR performed on serum could be useful to detect human herpesvirus-7 active infection in liver transplant recipients.

Diagnóstico da infecção ativa pelo herpesvirus humano-7 é difícil devido ao fato deste vírus ser ubíquo e poder causar infecção persistente no hospedeiro. O significado da detecção do DNA viral por reação em cadeia da polimerase não é claro e, reações cruzadas podem ocorrer em testes sorológicos. O objetivo deste estudo foi avaliar a nested-PCR para detectar infecção ativa pelo herpesvirus-7 em receptores hepáticos comparando com indivíduos sadios. Nested-PCR para herpesvirus-7 foi realizado em leucócitos e soro de 53 voluntários sadios e em soro de 29 receptores hepáticos. Nos voluntários sadios, herpesvirus-7 foi detectado em 28,3 por cento de leucócitos e 0 por cento de soro. herpesvirus-7 foi detectado em soro de 48,2 por cento de receptores hepáticos. Nested-PCR em DNA extraído de leucócitos detectou infecção latente e o estudo sugere que nested-PCR realizada em soro poderia ser útil para detectar infecção ativa por herpesvirus-7 em receptores de fígado.