High-resolution melt curve analysis for rapid detection of rifampicin resistance in Mycobacterium tuberculosis: a single-centre study in Iran

Arefzadeh, Samaneh; Azimi, Taher; Nasiri, Mohammad Javad; Nikpor, Zahra; Dabiri, Hossein; Doustdar, Farahnoosh; Goudarzi, Hossein; Allahyartorkaman, Mohammadreza

doi:10.1016/j.nmni.2020.100665

Cited by 14 publications

(8 citation statements)

References 30 publications

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“…A recent meta‐analysis evaluating performance of HRM in determining drug resistance in >4700 PTB isolates reported a sensitivity and specificity of 94% and 97%, respectively, for Rif and 93% and 98%, respectively, for Inh 12 . Such results could be attributable to resistance arising from genes outside those targeted in these studies, for example, 5% of RifR is attributable to mutations at sites other than the rpoB gene as reported earlier also 27 . Similarly, a review on gene loci responsible for InhR reported out of >5804 global isolates of InhR‐ M. tuberculosis , mutations in katG gene were found in 4271 (73.5%), followed by inhA‐15 (13.5%) and inhA‐8 (1.8%) 29 .…”

Section: Discussionsupporting

confidence: 60%

“…Prior studies have also reported high sensitivity and specificity for HRM, but there were false‐RifR and false‐RifS cases also. Arefzahde et al, 27 in the analysis on 80 PTB specimens, reported that the sensitivity and specificity of HRM for determining RifR was 100% and 89.3%, respectively, with false‐RifR reported in eight cases. Galarza et al 28 reported a sensitivity and specificity of 98.7% and 97.7%, respectively, for HRM in determining RifR in Peruvian PTB isolates.…”

Section: Discussionmentioning

confidence: 99%

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Diagnosing osteo‐articular tuberculosis and multidrug resistance using real‐time polymerase chain reaction and high‐resolution melt‐curve analysis

Sharma

et al. 2022

Journal Orthopaedic Research

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The study evaluated real-time quantitative polymerase chain reaction (qPCR) and high-resolution melt-curve analysis (HRM) for simultaneous diagnosis of osteoarticular tuberculosis (OATB) and drug resistance. Two hundred and fifty synovial fluid and pus specimens (20 confirmed OATB by culture, 130 suspected OATB, and 100 controls) processed in the Department of Medical Microbiology, PGIMER were subjected to qPCR using rpoB, MPB64, and IS6110 genes. All OATB positive specimens were subjected to HRM for detecting resistance to rifampicin and isoniazid. qPCR detected 129/150 OATB cases with a sensitivity of 86% (95% for confirmed and 84.6% for suspected OATB cases) and specificity of 100%. rpoB and MPB64 genes had higher sensitivity than IS6110 (86% vs. 74.6%). HRM reported eight multidrug resistant (MDR), two mono-rifampicin, and five mono-isoniazid resistant cases, all were concordant with gene sequencing. qPCR followed by HRM analysis offer a simple, accurate, and rapid platform for simultaneous detection of OATB and MDR.

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Section: Discussionsupporting

confidence: 60%

Section: Discussionmentioning

confidence: 99%

Diagnosing osteo‐articular tuberculosis and multidrug resistance using real‐time polymerase chain reaction and high‐resolution melt‐curve analysis

Sharma

et al. 2022

Journal Orthopaedic Research

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“…In the last few years, HRM PCR has become more widely applied for epidemiological purposes related to infectious diseases in addition to genotyping yeasts, viruses, or bacteria [23][24][25][26]. Indeed, this technique allows distinction of the different viral or bacterial strains [23][24][25], detection of mutated and wild-type strains [13], mutations located on resistance genes, such as resistance to rifampicin in Mycobacterium tuberculosis [27], or differentiation between Brucella species [28]. In parasitology, HRM analysis is extensively applied for protozoan parasites.…”

Section: Discussionmentioning

confidence: 99%

High-resolution melting analysis identifies reservoir hosts of zoonotic Leishmania parasites in Tunisia

et al. 2022

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Background Leishmaniasis is endemic in Tunisia and presents with different clinical forms, caused by the species Leishmania infantum, Leishmania major, and Leishmania tropica. The life cycle of Leishmania is complex and involves several phlebotomine sand fly vectors and mammalian reservoir hosts. The aim of this work is the development and evaluation of a high-resolution melting PCR (PCR-HRM) tool to detect and identify Leishmania parasites in wild and domestic hosts, constituting confirmed (dogs and Meriones rodents) or potential (hedgehogs) reservoirs in Tunisia. Methods Using in vitro-cultured Leishmania isolates, PCR-HRM reactions were developed targeting the 7SL RNA and HSP70 genes. Animals were captured or sampled in El Kef Governorate, North West Tunisia. DNA was extracted from the liver, spleen, kidney, and heart from hedgehogs (Atelerix algirus) (n = 3) and rodents (Meriones shawi) (n = 7) and from whole blood of dogs (n = 12) that did not present any symptoms of canine leishmaniasis. In total, 52 DNA samples were processed by PCR-HRM using both pairs of primers. Results The results showed melting curves enabling discrimination of the three Leishmania species present in Tunisia, and were further confirmed by Sanger sequencing. Application of PCR-HRM assays on reservoir host samples showed that overall among the examined samples, 45 were positive, while seven were negative, with no Leishmania infection. Meriones shawi were found infected with L. major, while dogs were infected with L. infantum. However, co-infections with L. major/L. infantum species were detected in four Meriones specimens and in all tested hedgehogs. In addition, multiple infections with the three Leishmania species were found in one hedgehog specimen. Sequence analyses of PCR-HRM products corroborated the Leishmania species found in analyzed samples. Conclusions The results of PCR-HRM assays applied to field specimens further support the possibility of hedgehogs as reservoir hosts of Leishmania. In addition, we showed their usefulness in the diagnosis of canine leishmaniasis, specifically in asymptomatic dogs, which will ensure a better evaluation of infection extent, thus improving elaboration of control programs. This PCR-HRM method is a robust and reliable tool for molecular detection and identification of Leishmania and can be easily implemented in epidemiological surveys in endemic regions. Graphical Abstract

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“…The leading cause of infertility is not clear, but it is assumed due to different risk factors, including smoking, alcohol consumption, changes in sexual behaviour, and even infectious diseases, meaningful increase in the prevalence rate of infertility can be observed. Among different infectious risk factors, the role of various viruses has attracted much attention (Abedi et al., 2018; Arefzadeh et al., 2020; Boeri et al., 2019; Garolla et al., 2013; Ghasemnejad et al., 2018; Khajehzadeh et al., 2018; Mahmoudi et al., 2019; Mousavi et al., 2020; Nasiri et al., 2020; Pormohammad et al., 2019; Pourentezari et al., 2016; Schuppe et al., 2017).…”

Section: Introductionmentioning

confidence: 99%

The evaluation of Human papilloma virus and human herpes viruses (EBV, CMV, VZV HSV‐1 and HSV‐2) in semen samples

et al. 2021

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There are a number of risk factors, especially viral diseases, which can lead to infertility. Among the various viral infections, much attention has been given to the role of the Papillomaviridae and Herpesviridae. After collecting 82 semen samples (37 teratospermia, 2 asthenozoospermia, 2 oligoasthenospermia, 1 oligospermia, 6 asthenoteratospermia and 34 normal semen samples), and washing them, the DNA from both freshly ejaculated spermatozoon and washed spermatozoa was extracted. Subsequently, the prevalence of EBV, CMV, HSV‐1, HSV‐2, VZV and HPV was evaluated using Multiplex PCR and Nested PCR. In this study, 1 normal and 5 abnormal semen samples were infected with HSV‐1 (1 normal, 4 teratospermia and 1 oligoasthenospermia). In addition, there were 2 VZV‐positive samples (both were teratozoospermia). Nested PCR indicated that 1 asthenozoospermia, 1 asthenoteratospermia, 3 teratospermia and 4 normal samples were HPV positive (including 8 HPV‐18 and 1 HPV‐33). Among 9 HPV‐positive subjects, 3 samples were negative after washing the infected samples. The prevalence of EBV, CMV, VZV, HSV‐1 and HSV‐2 remained unchanged prior to and after washing. Maybe sperm washing can be useful to eliminate HPV infection from semen samples, but further investigation is required because of the small number of samples.

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High-resolution melt curve analysis for rapid detection of rifampicin resistance in Mycobacterium tuberculosis: a single-centre study in Iran

Cited by 14 publications

References 30 publications

Diagnosing osteo‐articular tuberculosis and multidrug resistance using real‐time polymerase chain reaction and high‐resolution melt‐curve analysis

Diagnosing osteo‐articular tuberculosis and multidrug resistance using real‐time polymerase chain reaction and high‐resolution melt‐curve analysis

High-resolution melting analysis identifies reservoir hosts of zoonotic Leishmania parasites in Tunisia

The evaluation of Human papilloma virus and human herpes viruses (EBV, CMV, VZV HSV‐1 and HSV‐2) in semen samples

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