Evaluation of Real-Time and Conventional PCR Targeting Complex 85 Genes for Detection of Mycobacterium leprae DNA in Skin Biopsy Samples from Patients Diagnosed with Leprosy

ABSTRACT Leprosy is caused by Mycobacterium leprae, and it remains underdiagnosed in Burkina Faso. We investigated the use of fluorescent in situ hybridization (FISH) for detecting M. leprae in 27 skin samples (skin biopsy samples, slit skin samples, and skin lesion swabs) collected from 21 patients from Burkina Faso and three from Côte d’Ivoire who were suspected of having cutaneous leprosy. In all seven Ziehl-Neelsen-positive skin samples (four skin biopsy samples and three skin swabs collected from the same patient), FISH specifically identified M. leprae, including one FISH-positive skin biopsy sample that remained negative after testing with PCR targeting the rpoB gene and with the GenoType LepraeDR assay. Twenty other skin samples and three negative controls all remained negative for Ziehl-Neelsen staining, FISH, and rpoB PCR. These data indicate the usefulness of a microscopic examination of skin samples after FISH for first-line diagnosis of cutaneous leprosy. Accordingly, FISH represents a potentially useful point-of-care test for the diagnosis of cutaneous leprosy.

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Serological and Molecular Evidence of Brucella Species in the Rapidly Growing Pig Sector in Kenya

10.21203/rs.2.21914/v2 ◽

2020 ◽

Author(s):

James Miser Akoko ◽

Roger Pelle ◽

Velma Kivali ◽

Esther Schelling ◽

Gabriel Shirima ◽

...

Keyword(s):

Real Time ◽

Epidemiological Data ◽

Molecular Techniques ◽

Molecular Evidence ◽

Rt Pcr ◽

Conventional Pcr ◽

Brucella Infection ◽

Growing Pig ◽

Pcr Targeting

Abstract Background: Brucellosis is an emerging yet neglected zoonosis that has been reported in Kenya. Epidemiological data on brucellosis in ruminants is readily accessible; however, reports on brucellosis in pigs remain limited. This study sought to detect Brucella infection in pig serum by both serological and molecular techniques. Serum from 700 pigs randomly collected at a centralized abattoir in Nairobi region, Kenya were screened in parallel, using both Rose Bengal Test (RBT) and competitive Enzyme-Linked Immuno-sorbent Assay (cELISA) for antibodies against Brucella spp. All sera positive by RBT and 16 randomly selected negative samples were further tested using conventional PCR targeting bcsp31 geneand real-time PCR (RT-PCR) assays targeting IS711 and bcsp31 genes. Results: A prevalence of 0.57% (n = 4/700) was estimated using RBT; none of these samples was positive on cELISA. All RBT positive sera were also positive by both PCRs, while two sero-negative samples also tested positive on RT-PCR (n = 6/20). Brucella abortus was detected in four out of the six PCR positive samples through a real-time multiplex PCR. Conclusion: The detection of antibodies against Brucella spp. and DNA in serum from slaughterhouse pigs confirm the presence of Brucella in pigs. Therefore, investigation of the epidemiology and role of pigs in the transmission of brucellosis in Kenya is needed. Further targeted studies would be useful to systematically quantify and identify the spp. of Brucella in pigs.

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Detection of Mycobacterium leprae using real-time PCR in paucibacillary leprosy patients with negative acid-fast bacilli smears

Medical Journal of Indonesia ◽

10.13181/mji.v28i4.2643 ◽

2019 ◽

Vol 28 (4) ◽

pp. 351-7

Author(s):

Arleen Devita ◽

Fera Ibrahim ◽

Sri Linuwih Susetyo Wardhani Menaldi ◽

Angky Budianti ◽

Andi Yasmon

Keyword(s):

Skin Biopsy ◽

Real Time ◽

Skin Diseases ◽

False Negative ◽

Mycobacterium Leprae ◽

Skin Lesions ◽

Rt Pcr ◽

Pcr Assay ◽

Acid Fast Bacilli ◽

Diagnostic Capacity

BACKGROUND Leprosy is an infectious disease that is still a health problem worldwide, including in Indonesia. The clinical symptoms are similar to other skin diseases and it is difficult to establish a diagnosis for paucibacillary (PB) leprosy. Current serological and histopathological tests have limitations, especially in patients with negative acid-fast bacilli (AFB). Serological tests often give false-negative results, while histopathological results often consist of non-specific inflammation. Probe-based real-time polymerase chain reaction (RT-PCR) assays is an alternative test that may be more sensitive and more specific to detect Mycobacterium leprae. METHODS This study was done in June 2015 until March 2016; detected M. leprae in PB patients with negative AFB smears using TaqMan® probe-based RT-PCR assay on slit skin scrapings and skin biopsy specimens from 24 patients. The skin scrapings were obtained from skin tissue on ear lobes, skin lesions, as well as those from biopsy. Samples were tested with RT-PCR while histopathological examinations were only performed on skin from biopsy. RESULTS The RT-PCR assay showed positive results of 21%, 25%, and 96% for specimens obtained from skin scrapings of the ear lobe, skin lesions, and skin biopsy, respectively. On the other hand, the positive rate for the histopathological test from skin biopsy was 79%. It indicated that the TaqMan® RT-PCR assay could increase the diagnostic capacity of histopathological examination by as much as 17%. CONCLUSIONS TaqMan® PCR assay can improve the diagnostic capacity of histopathological examinations, which could be used as the new gold standard for the diagnosis of leprosy.

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Serological and molecular evidence of Brucella species in the rapidly growing pig sector in Kenya

10.21203/rs.2.21914/v3 ◽

2020 ◽

Author(s):

James Miser Akoko ◽

Roger Pelle ◽

Velma Kivali ◽

Esther Schelling ◽

Gabriel Shirima ◽

...

Keyword(s):

Real Time ◽

Epidemiological Data ◽

Molecular Techniques ◽

Molecular Evidence ◽

Brucella Species ◽

Rt Pcr ◽

Conventional Pcr ◽

Growing Pig ◽

Pcr Targeting

Abstract Background: Brucellosis is an emerging yet neglected zoonosis that has been reported in Kenya. Epidemiological data on brucellosis in ruminants is readily accessible; however, reports on brucellosis in pigs remain limited. This study sought to detect Brucella infection in pig serum by both serological and molecular techniques. Serum from 700 pigs randomly collected at a centralized abattoir in Nairobi region, Kenya were screened in parallel, using both Rose Bengal Test (RBT) and competitive Enzyme-Linked Immuno-sorbent Assay (cELISA) for antibodies against Brucella spp. All sera positive by RBT and 16 randomly selected negative samples were further tested using conventional PCR targeting bcsp31 gene and real-time PCR (RT-PCR) assays targeting IS711 and bcsp31 genes.Results: A prevalence of 0.57% (n = 4/700) was estimated using RBT; none of these samples was positive on cELISA. All RBT positive sera were also positive by both PCRs, while two sero-negative samples also tested positive on RT-PCR (n = 6/20). Brucella abortus was detected in four out of the six PCR positive samples through a real-time multiplex PCR.Conclusion: The detection of antibodies against Brucella spp. and DNA in serum from slaughterhouse pigs confirm the presence of Brucella in pigs. Therefore, investigation of the epidemiology and role of pigs in the transmission of brucellosis in Kenya is needed. Further targeted studies would be useful to systematically quantify and identify the spp. of Brucella in pigs.

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MULTIPLEX SYBR® GREEN-REAL TIME PCR (qPCR) ASSAY FOR THE DETECTION AND DIFFERENTIATION OF Bartonella henselae AND Bartonella clarridgeiae IN CATS

Revista do Instituto de Medicina Tropical de São Paulo ◽

10.1590/s0036-46652014000200001 ◽

2014 ◽

Vol 56 (2) ◽

pp. 93-95 ◽

Cited By ~ 9

Author(s):

Rodrigo Staggemeier ◽

Diogo André Pilger ◽

Fernando Rosado Spilki ◽

Vlademir Vicente Cantarelli

Keyword(s):

Real Time ◽

Bartonella Henselae ◽

Conventional Pcr ◽

Bartonella Species ◽

Chain Reaction ◽

Animal Shelters ◽

Blood Samples ◽

Two Samples ◽

Polymerase Chain ◽

Pcr Targeting

A novel SYBR® green-real time polymerase chain reaction (qPCR) was developed to detect two Bartonellaspecies, B. henselae and B. clarridgeiae, directly from blood samples. The test was used in blood samples obtained from cats living in animal shelters in Southern Brazil. Results were compared with those obtained by conventional PCR targeting Bartonella spp. Among the 47 samples analyzed, eight were positive using the conventional PCR and 12 were positive using qPCR. Importantly, the new qPCR detected the presence of both B. henselae and B. clarridgeiae in two samples. The results show that the qPCR described here may be a reliable tool for the screening and differentiation of two important Bartonella species.

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Comparative Value of Real Time and Conventional PCR in Antenatal Gender Determination

Bulletin of Egyptian Society for Physiological Sciences ◽

10.21608/besps.2016.8643 ◽

2016 ◽

Vol 36 (1) ◽

pp. 30-41

Author(s):

Naglaa Azab ◽

Rabab Salim ◽

Mohamed Farag

Keyword(s):

Real Time ◽

Conventional Pcr ◽

Gender Determination

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Molecular Characterization of Giardia duodenalis in Children and Adults Sampled in Algeria

Microorganisms ◽

10.3390/microorganisms9010054 ◽

2020 ◽

Vol 9 (1) ◽

pp. 54

Author(s):

Salem Belkessa ◽

Daniel Thomas-Lopez ◽

Karim Houali ◽

Farida Ghalmi ◽

Christen Rune Stensvold

Keyword(s):

Real Time ◽

Molecular Characterization ◽

Real Time Pcr ◽

Intestinal Parasites ◽

Giardia Duodenalis ◽

Specific Pcr ◽

Stool Samples ◽

Pcr Targeting ◽

Assemblage B

The molecular epidemiology of giardiasis in Africa remains unclear. A study was carried out across four hospitals in Algeria. A total of 119 fecal samples from 55 children, 37 adults, and 27 individuals of undetermined age, all scored positive for intestinal parasites by microscopy, and were screened by real-time PCR for Giardia. Molecular characterization of Giardia was performed by assemblage-specific PCR and PCR targeting the triose phosphate isomerase gene (tpi). Of the 119 samples, 80 (67%) were Giardia-positive by real-time PCR. For 48 moderately-highly real-time PCR-positive samples, tpi genotyping assigned 22 samples to Assemblage A and 26 to Assemblage B. Contrary to Assemblage A, Assemblage B exhibited substantial genetic diversity and allelic heterozygosity. Assemblage-specific PCR proved to be specific for discriminating Assemblage A or B but not as sensitive as tpi genotyping. We confirmed that real-time PCR is more sensitive than microscopy for detecting Giardia in stool samples and that robust amplification and sequencing of the tpi gene is feasible when moderate-to-strongly real-time PCR-positive samples are used. This study is one of the few performed in Africa providing genotyping data on Giardia infections in humans. Both assemblages A and B were commonly seen and not associated with specific sociodemographic data.

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Identification, Detection, and Enumeration of Human Bifidobacterium Species by PCR Targeting the Transaldolase Gene

Applied and Environmental Microbiology ◽

10.1128/aem.68.5.2420-2427.2002 ◽

2002 ◽

Vol 68 (5) ◽

pp. 2420-2427 ◽

Cited By ~ 126

Author(s):

Teresa Requena ◽

Jeremy Burton ◽

Takahiro Matsuki ◽

Karen Munro ◽

Mary Alice Simon ◽

...

Keyword(s):

Real Time ◽

16S Rdna ◽

Quantitative Pcr ◽

Gene Sequence ◽

Denaturing Gradient Gel Electrophoresis ◽

Bifidobacterium Longum ◽

Bifidobacterium Adolescentis ◽

Fecal Samples ◽

Real Time Quantitative Pcr ◽

Pcr Targeting

ABSTRACT Methods that enabled the identification, detection, and enumeration of Bifidobacterium species by PCR targeting the transaldolase gene were tested. Bifidobacterial species isolated from the feces of human adults and babies were identified by PCR amplification of a 301-bp transaldolase gene sequence and comparison of the relative migrations of the DNA fragments in denaturing gradient gel electrophoresis (DGGE). Two subtypes of Bifidobacterium longum, five subtypes of Bifidobacterium adolescentis, and two subtypes of Bifidobacterium pseudocatenulatum could be differentiated using PCR-DGGE. Bifidobacterium angulatum and B. catenulatum type cultures could not be differentiated from each other. Bifidobacterial species were also detected directly in fecal samples by this combination of PCR and DGGE. The number of species detected was less than that detected by PCR using species-specific primers targeting 16S ribosomal DNA (rDNA). Real-time quantitative PCR targeting a 110-bp transaldolase gene sequence was used to enumerate bifidobacteria in fecal samples. Real-time quantitative PCR measurements of bifidobacteria in fecal samples from adults correlated well with results obtained by culture when either a 16S rDNA sequence or the transaldolase gene sequence was targeted. In the case of samples from infants, 16S rDNA-targeted PCR was superior to PCR targeting the transaldolase gene for the quantification of bifidobacterial populations.

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