Fluorescence In Situ Hybridization (FISH) Assays for Diagnosing Malaria in Endemic Areas

Shah, Jyotsna; Mark, Olivia; Weltman, Helena; Barceló, Nicolás E.; Lo, Wai Hung; Wrońska, Danuta; Kakkilaya, Srinivas B.; Rao, A V Prabhakara; Bhat, Shivananda K; Sinha, Ruchi; Omar, Sabah A.; Obare, Peter; Moro, Manuel; Gilman, Robert H.; Harris, N.

doi:10.1371/journal.pone.0136726

Cited by 23 publications

(63 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Despite the fact that PCR was used, which is a more sensitive method for evaluating diagnostic performance, the sensitivity of the FISH assay was poor in this study as compared to a previous study by Shah et al which showed that the sensitivity of the P-Genus FISH assay was 98% when compared to Giemsa microscopy [7]. The specificity of P-Genus FISH in the current study was 75.8% which is closely similar to the previous study (83.4%) by Shah et al although she compared her study with Giemsa microscopy.…”

Section: Discussioncontrasting

confidence: 58%

Comparative evaluation of fluorescent in situ hybridization and Giemsa microscopy with quantitative real-time PCR technique in detecting malaria parasites in a holoendemic region of Kenya

Osoga

Waitumbi

Guyah

et al. 2017

Malar J

View full text Add to dashboard Cite

BackgroundEarly and accurate diagnosis of malaria is important in treatment as well as in the clinical evaluation of drugs and vaccines. Evaluation of Giemsa-stained smears remains the gold standard for malaria diagnosis, although diagnostic errors and potential bias estimates of protective efficacy have been reported in practice. Plasmodium genus fluorescent in situ hybridization (P-Genus FISH) is a microscopy-based method that uses fluorescent labelled oligonucleotide probes targeted to pathogen specific ribosomal RNA fragments to detect malaria parasites in whole blood. This study sought to evaluate the diagnostic performance of P-Genus FISH alongside Giemsa microscopy compared to quantitative reverse transcription polymerase chain reaction (qRT-PCR) in a clinical setting.MethodFive hundred study participants were recruited prospectively and screened for Plasmodium parasites by P-Genus FISH assay, and Giemsa microscopy. The microscopic methods were performed by two trained personnel and were blinded, and if the results were discordant a third reading was performed as a tie breaker. The diagnostic performance of both methods was evaluated against qRT-PCR as a more sensitive method.ResultsThe number of Plasmodium positive cases was 26.8% by P-Genus FISH, 33.2% by Giemsa microscopy, and 51.2% by qRT-PCR. The three methods had 46.8% concordant results with 61 positive cases and 173 negative cases. Compared to qRT-PCR the sensitivity and specificity of P-Genus FISH assay was 29.3 and 75.8%, respectively, while microscopy had 58.2 and 93.0% respectively. Microscopy had a higher positive and negative predictive values (89.8 and 68.0% respectively) compared to P-Genus FISH (56.0 and 50.5%). In overall, microscopy had a good measure of agreement (76%, k = 0.51) compared to P-Genus FISH (52%, k = 0.05).ConclusionThe diagnostic performance of P-Genus FISH was shown to be inferior to Giemsa microscopy in the clinical samples. This hinders the possible application of the method in the field despite the many advantages of the method especially diagnosis of low parasite density infections. The P-Genus assay has great potential but application of the method in clinical setting would rely on extensive training of microscopist and continuous proficiency testing.

show abstract

Section: Discussioncontrasting

confidence: 58%

Comparative evaluation of fluorescent in situ hybridization and Giemsa microscopy with quantitative real-time PCR technique in detecting malaria parasites in a holoendemic region of Kenya

Osoga

Waitumbi

Guyah

et al. 2017

Malar J

View full text Add to dashboard Cite

show abstract

“…While we used nucleic acid stains for sensitive detection of P. falciparum parasites in thin blood smear, different probes that are specific to a set of pathogens can also be utilized. The past decades has seen much development of pathogen specific probes [75, 76, 77, 78]. Being low-cost and highly configurable, Octopi has the potential to help realize the wide spread use of these new probes in field diagnostics.…”

Section: Discussionmentioning

confidence: 99%

Octopi: Open configurable high-throughput imaging platform for infectious disease diagnosis in the field

Soto-Montoya

Voisin

et al. 2019

Preprint

View full text Add to dashboard Cite

Access to quantitative, robust, yet affordable diagnostic tools is necessary to reduce global infectious disease burden. Manual microscopy has served as a bedrock for diagnostics with wide adaptability, although at a cost of tedious labor and human errors. Automated robotic microscopes are poised to enable a new era of smart field microscopy but current platforms remain cost prohibitive and largely inflexible, especially for resource poor and field settings. Here we present Octopi, a low-cost ($250-$500) and reconfigurable autonomous microscopy platform capable of automated slide scanning and correlated bright-field and fluorescence imaging. Being highly modular, it also provides a framework for new disease-specific modules to be developed.We demonstrate the power of the platform by applying it to automated detection of malaria parasites in blood smears. Specifically, we discovered a spectral shift on the order of 10 nm for DAPI-stained Plasmodium falciparum malaria parasites. This shift allowed us to detect the parasites with a low magnification (equivalent to 10x) large field of view (2.56 mm 2 ) module.Combined with automated slide scanning, real time computer vision and machine learningbased classification, Octopi is able to screen more than 1.5 million red blood cells per minute for parasitemia quantification, with estimated diagnostic sensitivity and specificity exceeding 90% at parasitemia of 50/ul and 100% for parasitemia higher than 150/l. With different modules, we further showed imaging of tissue slice and sputum sample on the platform. With roughly two orders of magnitude in cost reduction, Octopi opens up the possibility of a large robotic microscope network for improved disease diagnosis while providing an avenue for collective efforts for development of modular instruments.One sentence summary: We developed a low-cost ($250-$500) automated imaging platform that can quantify malaria parasitemia by scanning 1.5 million red blood cells per minute.Lack of cost-effective diagnostics is a major hurdle in global fight against infectious disease, 2 specially in resource poor settings [1]. This leaves our world in a highly vulnerable position 3 with therapeutic drugs being either overused, leading to drug resistant strains or not acces-4 sible to people who actually need these treatments [2]. Since health care is delivered around 5 the world in a tiered structure, local context such as high cost, lack of trained personal or 6 low throughput of many available diagnostics tests plays a large detrimental role on quality 7 of delivered health-care [3]. 8 Because of the versatility and wide adoption of manual microscopy [4] and its role in 9 direct visual identification of parasites [5], it remains a WHO gold standard for numerous 10 diseases [1]. Despite technological advancements in related fields, the practice of conventional 11 manual microscopy has remained largely unchanged over the last half century and suffers 12 from several drawbacks. With an average lab technician spending 6 to 8 hours imaging and 13 ...

show abstract

“…The advantages of FISH assays for malaria compared to the more sensitive nested PCR assays have been previously discussed in detail for the P. falciparum and P. vivax FISH assays [10], and the same considerations also apply to the PK-FISH assay. In essence, the FISH assays require no specialized equipment other than a light microscope with a LED source and filter attachments, and are less costly and simpler to perform than PCR-based tests.…”

Section: Discussionmentioning

confidence: 99%

A dual colour fluorescence in situ hybridization (FISH) assay for identifying the zoonotic malaria parasite Plasmodium knowlesi with a potential application for the specific diagnosis of knowlesi malaria in peripheral-level laboratories of Southeast Asia

et al. 2017

Self Cite

View full text Add to dashboard Cite

Background Plasmodium knowlesi is primarily responsible for zoonotic malaria in several Southeast Asian countries. Precise identification of the parasite in the blood of patients presently relies on an expensive and elaborate PCR procedure because microscopic examination of blood and other available field identification techniques lack adequate specificity. Therefore, the use of a simple and inexpensive dual-colour fluorescence in situ hybridization (FISH) assay, analogous to FISH assays recently described for Plasmodium falciparum and Plasmodium vivax, was investigated as a potential tool for identifying P. knowlesi.ResultsA P. knowlesi 18S rDNA sequence-based DNA probe was used to test thin blood smears of P. knowlesi by FISH, and fluorescence viewed in a light microscope fitted with a light emitting diode light source and appropriate emission and barrier filters. The limit of detection in the P. knowlesi FISH assay was 84 parasites per μl in infected monkey blood and 61 parasites per μl for P. knowlesi cultured in human blood. The P. knowlesi-specific FISH probe detected only P. knowlesi and not P. falciparum, Plasmodium malariae, Plasmodium ovale, P. vivax or a panel of other human blood-borne pathogens. A previously described Plasmodium genus-specific probe used simultaneously in the P. knowlesi FISH assay reacted with all tested Plasmodium species.ConclusionsTo our knowledge, this is the first description of a FISH assay for P. knowlesi that is potentially useful for diagnosing infections in remote laboratories in endemic countries.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-017-2273-7) contains supplementary material, which is available to authorized users.

show abstract

Fluorescence In Situ Hybridization (FISH) Assays for Diagnosing Malaria in Endemic Areas

Cited by 23 publications

References 20 publications

Comparative evaluation of fluorescent in situ hybridization and Giemsa microscopy with quantitative real-time PCR technique in detecting malaria parasites in a holoendemic region of Kenya

Comparative evaluation of fluorescent in situ hybridization and Giemsa microscopy with quantitative real-time PCR technique in detecting malaria parasites in a holoendemic region of Kenya

Octopi: Open configurable high-throughput imaging platform for infectious disease diagnosis in the field

A dual colour fluorescence in situ hybridization (FISH) assay for identifying the zoonotic malaria parasite Plasmodium knowlesi with a potential application for the specific diagnosis of knowlesi malaria in peripheral-level laboratories of Southeast Asia

Contact Info

Product

Resources

About