A survey of bancroftian filariasis among South-East Asian expatriate workers in Saudi Arabia

In a survey of bancroftian filariasis among expatriate workers from five South-East Asian countries (India, Bangladesh, Sri Lanka, Thailand and the Philippines) where human filariasis is endemic, 762 individuals were examined in the Abha area (Asir) of south-western Saudi Arabia. A prevalence of microfilaraemia of 3.5% and a mean mf density of 6.0/20 mm³of blood was found among 259 Indian males only. In three out of 9 microfilaraemic cases, clinical signs suggestive of filariasis were observed. The only species identified was Wuchereria bancrofti showing strict nocturnal periodicity. Preliminary laboratory studies on the susceptibility of local mosquitoes to infection with W. bancrofti in which laboratory-bred Culex (C.) pipiens was fed directly on a microfilaraemic volunteer from Madras, South India, revealed that this species was highly susceptible to the Madras strain of the parasite with an average infection rate of 57 (range 41–75)% and a worm burden of 3.5 L³/infective mosquito. This is the first report that local Cx. pipiens mosquitoes may act as a potential vector of introduced bancroftian filariasis in Saudi Arabia. The potential danger of bancroftian filariasis importation and, more importantly, the establishment of new self-sustained foci of the disease is likely to depend upon the abundance of mf carriers and density of vector mosquitoes feeding on carriers.     

A new multiplex real-time polymerase chain reaction assay for the diagnosis of periprosthetic joint infection

Objectives: A new multiplex real-time polymerase chain reaction (PCR) assay was developed to detect methicillin-resistant Staphylococcus (MRS) and to distinguish between gram-positive and gram-negative bacteria. In this study, we validated the sensitivity and specificity of this assay with periprosthetic joint infections (PJIs) and evaluated the utility of PCR for culture-negative PJI.

Methods: Forty-five samples from 23 infectious PJI cases and 106 samples from 64 non-infectious control cases were analyzed by real-time PCR using a LightCycler Nano^® system. Twenty-eight clinical samples, comprising bacteria of known species isolated consecutively in the microbiological laboratory of our hospital, were used to determine the spectrum of bacterial species that could be detected using the new multiplex primers and probes.

Results: The sensitivity and specificity of the MRS- and universal-PCR assays were 92% and 99%, and 91% and 88%, respectively. Twenty-eight species of clinically isolated bacteria were detected using this method and the concordance rate for the identification of gram-positive or gram-negative organisms was 96%. Eight samples were identified as PCR-positive despite a culture-negative result.

Conclusion: This novel multiplex real-time PCR system has acceptable sensitivity and specificity and several advantages; therefore, it has potential use for the diagnosis of PJIs, particularly in culture-negative cases.     

等位基因特异性多重聚合酶链反应方法用于快速检测结核分枝杆菌利福平耐药株

目的建立检测耐利福平(RFP)结核分枝杆菌的等位基因特异性多重聚合酶链反应(multiplex allele specific polymerase chain reaction,MAS-PCR)方法,快速、特异地检测rpoB基因核心突变区的主要突变,用于快速诊断结核分枝杆菌对利福平的耐药性。方法根据结核分枝杆菌的rpoB序列,分别设计出3对特异性寡聚核苷酸引物,采用MAS-PCR技术,分别检测rpoB基因上531、526、516这3个最常见的突变位点的突变。结果对临床分离的利福平敏感株(15株)及利福平突变株(81株)进行检测,以直接测序结果为参照,对利福平耐药株的总检出率为81.5%(66/81)。结论MAS-PCR方法敏感、特异,可快速、简便地检测结核分枝杆菌rpoB基因突变,有利于耐药结核分枝杆菌的快速检测。     

rDNA-ITS2 polymerase chain reaction assay for the sibling species of Anopheles fluviatilis

Species-specific differences in the nucleotide sequences of the second internal transcribed spacer (ITS2) region of the ribosomal DNA (rDNA) were used to develop a diagnostic polymerase chain reaction (PCR) assay for two of the sibling species of the Anopheles fluviatilis complex, members of which are major vectors of malaria in central and northern parts of India. This assay consisted of a three primer reaction, which could amplify the DNA of both the species producing fragments of two distinct sizes, 350 bp for species X and 450 bp for species Y, respectively. The assay was found to be highly specific and sensitive.     

A pentaplex real-time polymerase chain reaction assay for detection of four species of soil-transmitted helminths

Soil-transmitted helminth infections remain a major public health burden in low- and middle-income countries. The traditional diagnosis by microscopic examination of fecal samples is insensitive and time-consuming. In this study, a pentaplex real-time polymerase chain reaction (PCR) was evaluated for the simultaneous detection of Ancylostoma, Necator americanus, Ascaris lumbricoides, and Strongyloides stercoralis. The results were compared with those obtained by conventional parasitological diagnostic methods. Real-time PCR was positive in 48 of 77 samples (62.3%) and microscopic examination was positive in six samples (7.8%) only (P < 0.05). In conclusion, the real-time PCR assay described in this study provides a specific and sensitive diagnostic tool for the detection of these four helminth species in epidemiological studies and monitoring of treatment programs.     

A specific and sensitive assay for the Lyme disease spirochete Borrelia burgdorferi using the polymerase chain reaction

A highly specific and sensitive assay for Borrelia burgdorferi, the causative agent of Lyme disease, was developed using the polymerase chain reaction (PCR). The target DNA sequence was of chromosomal origin and conserved, by hybridization analyses, among all strains of B. burgdorferi tested but was not present in the most closely related member of the genus, B. hermsii. The PCR assay developed from this sequence reacted with 17 of 18 strains of B. burgdorferi but not with any other Borrelia species tested. The assay was sensitive to fewer than five copies of the B. burgdorferi genome, even in the presence of a 10(6)-fold excess of eukaryotic DNA. This assay should greatly facilitate the accurate diagnosis of Lyme disease and provide a means with which to investigate the pathogenesis, transmission, and basic biology of B. burgdorferi.     

A genus- and species-specific nested polymerase chain reaction malaria detection assay for epidemiologic studies.

A nested polymerase chain reaction (PCR) assay that uses Plasmodium genus-specific primers for the initial PCR (nest 1) amplification and either genus- or species-specific primers for the nest 2 amplifications was tested on laboratory and field samples. With in vitro cultured Plasmodium falciparum-infected blood samples, it was capable of detecting six parasites/microl of blood using DNA prepared from 25-microl blood spots on filter paper. The assay was evaluated on fingerprick blood samples collected on filter paper from 129 individuals living in a malaria-endemic area in Malaysia. Malaria prevalence by genus-specific nested PCR was 35.6% (46 of 129) compared with 28.7% (37 of 129) by microscopy. The nested PCR detected seven more malaria samples than microscopy in the first round of microscopic examination, malaria in three microscopically negative samples, six double infections identified as single infections by microscopy and one triple infection identified as a double infection by microscopy. The nested PCR assay described is a sensitive technique for collecting accurate malaria epidemiologic data. When coupled with simple blood spot sampling, it is particularly useful for screening communities in remote regions of the world.     

Development of a quantitative real-time polymerase chain reaction assay specific for Orientia tsutsugamushi

Two specific and sensitive polymerase chain reaction (PCR) assays were developed to detect and quantitate Orientia tsutsugamushi, the agent of scrub typhus, using a portion of the 47-kD outer membrane protein antigen/ high temperature requirement A gene as the target. A selected 47-kD protein gene primer pair amplified a 118-basepair fragment from all 26 strains of O. tsutsugamushi evaluated, but it did not produce amplicons when 17 Rickettsia and 18 less-related bacterial nucleic acid extracts were tested. Similar agent specificity for the real-time PCR assay, which used the same primers and a 31-basepair fluorescent probe, was demonstrated. This sensitive and quantitative assay determination of the content of O. tsutsugamushi nucleic acid used a plasmid containing the entire 47-kD gene from the Kato strain as a standard. Enumeration of the copies of O. tsutsugamushi DNA extracted from infected tissues from mice and monkeys following experimental infection with Orientia showed 27-5552 copies/microL of mouse blood, 14448-86012 copies/microL of mouse liver/spleen homogenate, and 3-21 copies/microL of monkey blood.     

Multiplex polymerase chain reaction assay for selective detection of Salmonella enterica serovar typhimurium

A multiplex polymerase chain reaction (PCR) assay was developed for the identification of Salmonella enterica serovar Typhimurium. Three sets of primers were designed for detecting O4, H:i, and H:1,2 antigen genes from the antigen-specific genes rfbJ, fliC, and fljB, respectively. These were evaluated in a multiplex PCR assay by using DNAs from S. enterica serovar Typhimurium, 15 other Salmonella serovars, and 8 non-Salmonella enteric pathogens. Multiplex PCR proved to be capable of identifying S. enterica serovar Typhimurium specifically and differentiating it from other Salmonella serovars in addition to non-Salmonella enteric pathogens. Thus, this multiplex PCR assay can be practically applied to the identification of S. enterica serovar Typhimurium.     

荧光定量PCR检测嗜吞噬细胞无形体

目的采用新型TaqMan-MGB探针建立检测嗜吞噬细胞无形体的实时荧光定量PCR(quantitativereal-timePCR)方法。方法依据gltA基因序列设计嗜吞噬细胞无形体特异引物和探针,以克隆的嗜吞噬细胞无形体gltA基因片段作DNA模板,在荧光定量PCR检测仪(ABI7900HT)上建立实时荧光定量检测方法。结果建立的定量标准曲线的循环阈值(Ct)与模板拷贝数呈良好的线性关系(r=0.996);与套式PCR相比较,荧光定量PCR检测的灵敏度是其100倍。用荧光定量PCR检测其它相关立克次体和细菌DNA样本,检出结果几乎为0;对荧光定量PCR检测重复性进行分析,变异系数(CV)批内和批间误差在0~2.1%之间,证明该荧光定量PCR具有种特异性和良好的重复性。用荧光定量PCR检测体疑染嗜吞噬细胞无形体的10份蜱和30份小鼠脾脏标本,结果与套式PCR检测结果有密切相关性,但是定量PCR检测敏感性和准确率均高于套式PCR。结论本研究建立的检测嗜吞噬细胞无形体的实时荧光定量PCR具有很高的特异性和敏感性,特别适合检出样本中微量嗜吞噬细胞无形体。     

布鲁氏菌PCR鉴定方法的研究与应用

目的采用PCR、PCR SSCP方法对布鲁氏菌进行快速鉴定,并对其种、型鉴别。方法分析已发表布鲁氏菌属、种、型基因序列,寻找出不 同布鲁氏菌的种、型特异性碱基分布规律,设计特异性引物,用于布鲁氏菌属、种、型的鉴定;根据聚合酶链反应 单链构象多态性（PCR SSCP）分析原理,建立PCR SSCP方法,用于区分牛种A19疫苗株与其他型布鲁氏菌。结果所建立的PCR方法能高效、准确鉴定出布鲁氏菌,并能 对其种、型进行区分;PCR SSCP方法可将A19疫苗株从其他型布鲁氏菌中区分出来。结论利用PCR、PCR SSCP方法能快速、准确地进行布鲁氏 菌属、种、型以及疫苗株A19的鉴别,且简便、可靠,便于临床应用。     

Real-time polymerase chain reaction assay for cell-associated HTLV type I DNA viral load

We have developed a quantitative real-time PCR assay for HTLV-I DNA. This assay approach uses real-time monitoring of fluorescent signal generation as a consequence of Taq-mediated amplification of specific target sequences to allow real-time kinetic analysis of amplicon production. This kinetic approach yields excellent sensitivity and an extremely broad linear dynamic range, and ensures that quantitation is based on analysis during the exponential phase of amplification, regardless of the input template copy number. The HTLV-I DNA assay has a nominal threshold sensitivity of 10 copy Eq/reaction, although single-copy plasmid template can be detected at frequencies consistent with statistical prediction. The linear dynamic range is in excess of 5 logs. Interassay reproducibility averages 14% (coefficient of variation) for control templates over a range of 10(1) to 10(6) copy Eq/reaction and 25%, based on studies of extraction and analysis of replicate aliquots of PBMC specimens from HTLV-I-infected subjects. The primer/probe combination targets tax sequences conserved across described HTLV-I and HTLV-II isolates. Parallel quantitation in the same samples of an endogenous sequence present at a known copy number per cell allows normalization of results for potential variation in DNA recovery. Availability of this assay should facilitate studies of basic pathogenesis and clinical evaluation of HTLV-I and HTLV-II infection, as well as assessment of therapeutic approaches.     

Invasive pulmonary aspergillosis diagnosed early by polymerase chain reaction assay.

We compared the usefulness of a polymerase chain reaction (PCR) assay for the early diagnosis of invasive pulmonary aspergillosis with the serodiagnosis of sufficient concentrations of galactomannan using the same serum samples. A patient was treated with prednisolone for the management of hepatitis. Computed tomography (CT) scan of the chest showed the nodular shadow with a cavity containing a clear fungus ball. DNA of Aspergillus spp. from a serum sample was detected and using the same serum sample, both latex agglutination and sandwich enzyme-linked immunosorbent assay (ELISA) of galactomannan were negative. PCR assay provides an early diagnosis of invasive pulmonary aspergillosis compared with ELISA of galactomannan.     

A monoclonal antibody-based enzyme immunoassay for detecting parasite antigenemia in bancroftian filariasis

We evaluated a monoclonal antibody-based enzyme immunoassay for detecting soluble parasite antigen in sera collected in an area in South India endemic for Wuchereria bancrofti. Filarial antigen was detected in sera from 56 of 57 microfilaremic patients, 9 of 64 aminofilaremic patients with clinical filariasis, and 11 of 70 endemic controls. Antigen was not detected in sera from patients from nonendemic areas who had a variety of other filarial and nonfilarial helminth infections. Parasite antigen titers were significantly correlated with microfilarial counts in night blood smears (r = .64, P less than .01). Negative antigen tests in patients with clinical filariasis may be explained in part by antibody-mediated clearance of circulating antigen. Antibodies to circulating W. bancrofti antigen were detected in 41 of 55 antigen-negative sera from patients with clinical filariasis. Despite this limitation, detecting parasite antigen by enzyme immunoassay provides significant advantages over previously available methods for diagnosing active W. bancrofti infection.     

JH probe real-time quantitative polymerase chain reaction assay for Bcl-2/IgH rearrangements

Follicular lymphoma (FL) characteristically bears the t(14;18)(q32;q21). However, only approximately 75% of the consequent Bcl-2 breakpoints lie within the major breakpoint region (MBR) or the minor cluster region (mcr). While these can be quantified by cluster region-specific real-time quantitative polymerase chain reaction (RQ-PCR), a significant proportion of cases are left requiring a customized approach. Therefore, an RQ-PCR assay for the quantification of Bcl-2/IgH breakpoints has been developed that uses germline JH TaqMan probes and germline JH primers in combination with customized forward primers. Validation of this approach by comparison with an established MBR RQ-PCR showed both techniques to be concordant across a wide range of copy numbers with a sensitivity of five copies per 10(5) cells. In addition, to generate standard curves equating to diverse Bcl-2/IgH rearrangements, a strategy for using placental DNA as a surrogate standard was devised. The performance of the assay in detecting molecular evidence of disease in sequential biopsies from five patients (three with atypical Bcl-2/IgH breakpoints identified by long-range or inverse PCR, one MBR+ and one mcr+) was tested. This alternative approach represents a sensitive and specific means of quantifying common and atypical Bcl-2/IgH rearrangements and maximizes the number of patients with FL suitable for molecular monitoring.     

Diagnostic application of a polymerase chain reaction assay for the Whipple's disease bacterium to intestinal biopsies

BACKGROUND & AIMS: The uncultured Whipple's disease bacterium (Tropheryma whippelii) was characterized in 1991-1992 by polymerase chain reaction (PCR) and sequencing of the bacterial 16S ribosomal RNA gene. The aim of this study was to develop a PCR assay for diagnostic purposes. METHODS: Modified primers for PCR and a specific probe for hybridization were designed. The specificity of this PCR assay was tested using 37 bacterial control strains and intestinal biopsy samples from 16 patients without Whipple's disease. The sensitivity was tested in 88 intestinal biopsy samples from 35 patients with Whipple's disease. RESULTS: PCR and hybridization were negative in all 37 bacterial controls and in all 16 patients without Whipple's disease. Before therapy, DNA of T. whippelii was detected in all 30 patients with Whipple's disease from whom formalin-fixed biopsy material was available, whereas Bouin-fixed material was negative. During and after treatment, PCR was negative in 23 of the 24 patients who were followed up. Generally, conversion to negative occurred within 1 year. Despite negative intestinal PCR, symptomatic cerebral Whipple's disease appeared in 3 patients. CONCLUSIONS: This PCR assay is specific and sensitive and is applicable as a diagnostic test. However, PCR from intestinal biopsy samples seems less helpful for monitoring the effect of treatment. (Gastroenterology 1996 Jun;110(6):1735-43)