PCR based Malaria diagnostics : – Method development and application

Background Almost half of the world’s population is at risk of malaria. Malaria is a parasitic vector borne disease spread by infected female Anopheles mosquitos. The protozoan parasite belongs to the genus Plasmodium group. In humans five main species are described, P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. The recent years the WHO has reported that over 200 million malaria cases, and 400 000 deaths occur every year. Microscopy and rapid diagnostic tests (RDTs) are used in routine malaria diagnostics, but these methods have their challenges in accurate sensitivity and specificity. Aim To develop a new robust and user-friendly PCR method, highly sensitive and specific, and to assess and apply this method on relevant clinical material. Methods We developed a new single-step genus-specific conventional PCR targeting the cytochrome b gene (cytb) on the mitochondrial genome, and modified a species-specific conventional PCR targeting the chromosomal small subunit ribosomal RNA 18S locus. The methods were assessed and compared with routine microscopy and a gold standard nested 18S PCR using a Norwegian clinical collection (N=135), before applied in a multi-centre hospital-based study in India (N=1412). We also converted the conventional genus-specific cytb PCR to two real-time PCR assays applying SYBR Green and a TaqMan probe. The two assays were assessed using reference material, the Norwegian material (N=113) and a Tanzanian P. falciparum field collection (N= 74), and compared to five other relevant real-time PCR methods. Sequencing of the genus-specific cytb PCR products was also performed. Results Among the Norwegian sample collection (N=135) the novel genus-specific cytb PCR had a 100% sensitivity (28/28), nested 18S PCR 96% (27/28), and microscopy 93% (26/28). The 28 positive cytb PCR products were sequenced revealing six single nucleotide polymorphisms (SNPs) and one insert/deletion allowing for species determination of the 28 sequences. In the Indian multi-centre study, the cytb PCR found a malaria prevalence of 19% (268/1412). Overall, 46% P. falciparum and 38% P. vivax single infections were detected, while P. falciparum and P. vivax double infection was found in 11%, and P. malariae in 5% of the admitted fever patients. Routine microscopy and RDT had a low sensitivity compared to PCR, 29% and 24% respectively. Furthermore, the conventional cytb PCR was successfully converted to real-time PCR. The SYBR assay showed a higher sensitivity and specificity than using a probe. Focusing on detecting low-level parasitaemia, SYBR was also more beneficial to apply than probe due to melting curve analysis (MCA) can reveal unspecific binding or primer-dimers. Compared to the five other relevant real-time PCR methods our cytb SYBR PCR was the most sensitive genus-specific method. Conclusion Choice of amplification target is relevant for achieving high sensitivity in detecting low-level parasitaemia, and it is advantageous to use an intercalating fluorescence dye suitable for MCA. The highly sensitive, specific and user-friendly cytb SYBR PCR can be a useful tool in epidemiological and surveillance research, as well as for clinical malaria diagnostics. The method is genus-specific, which is an advantage in large screening projects. Among ambiguous samples, and in settings where species-specific PCR fails to detect low-level parasitaemia, confirmation and species identification by sequencing of the genus-specific real-time PCR products can be used as a contingency

Detection of remaining Plasmodium DNA and gametocytes during follow up after curative malaria treatment among returned travellers in Norway

Background PCR can be positive weeks after effective malaria treatment, potentially leading to over diagnose of recrudescence and re-infections. The DNA detected by PCR post-treatment might stem from residuals of destroyed asexual parasites, or from live gametocytes. The objective of this clinical observational study was to describe the presence of positive PCR for Plasmodium falciparum and Plasmodium vivax in follow-up samples post-treatment from returned travellers, and the proportion of positive PCR due to gametocytes. Methods Whole blood was collected during hospitalization and outpatient routine follow-up from 13 patients with imported malaria. DNA was extracted applying QIAamp DNA Blood Mini Kit, while mRNA was collected and extracted applying PAXgene Blood RNA Tubes and Kit. All DNA samples (N = 25) were analysed with a genus-specific cytb real-time SYBR PCR, and P. falciparum DNA samples (N = 22) were also analysed with a falciparum-specific varATS real-time TaqMan PCR. All the mRNA samples (N = 18) were analysed with both a genus-specific 18S rRNA RT-PCR and a gametocyte-specific Pfs25 (P. falciparum)/Pvs25 (P. vivax) RT-PCR. Results Latest samples were collected at day 1 (n = 2) and from day 11–54 (n = 11) after treatment. Genus DNA cytb PCR was positive up to 49 days after effective treatment, and 18S rRNA transcripts from active P. falciparum parasites were detectable for at least 11 days. Gametocyte-specific mRNA was detected at latest only two days after treatment. Among six patients with late positive PCR for P. falciparum, four had high parasitaemia at admittance (6–30%), while two had parasitaemia < 2%. Late detection of P. vivax was not found by any of the PCR methods. Conclusions DNA-based PCR can be positive up to at least seven weeks after curative malaria treatment, potentially leading to over-diagnose of recrudescence and re-infections. Based on the observations in this study, it is unclear if the DNA origins from residuals of destroyed parasites or live gametocytes, warranting further investigations.publishedVersio

Malaria prevalence and performance of diagnostic tests among patients hospitalized with acute undifferentiated fever in Zanzibar

Background: Control efforts in Zanzibar reduced the burden of malaria substantially from 2000 to 2015, but re-emergence of falciparum malaria has been observed lately. This study evaluated the prevalence of malaria and performance of routine diagnostic tests among hospitalized fever patients in a 1.5 years period in 2015 and 2016. Methods: From March 2015 to October 2016, paediatric and adult patients hospitalized with acute undifferentiated fever at Mnazi Mmoja Hospital, Zanzibar were included. The malaria prevalence, and performance of rapid diagnostic test (RDT) and microscopy, were assessed using polymerase chain reaction (PCR) as gold standard. Results: The malaria prevalence was 9% (63/731). Children under 5 years old had lower malaria prevalence (5%, 14/260) than older children (15%, 20/131, p = 0.001) and persons aged 16 to 30 years (13%, 15/119, p = 0.02), but not different from persons over 30 years old (6%, 14/217, p = 0.7). All cases had Plasmodium falciparum infection, except for one case of Plasmodium ovale. Ten malaria patients had no history of visiting mainland Tanzania. The RDT had a sensitivity of 64% (36/56) and a specificity of 98% (561/575), and microscopy had a sensitivity of 50% (18/36) and a specificity of 99% (251/254), compared to PCR. The malaria parasitaemia was lower in patients with false negative results on RDT (median 7 × 103 copies/µL, interquartile range [IQR] 2 × 103 – 8 × 104, p = 0.002) and microscopy (median 9 × 103 copies/µL, IQR 8 × 102 – 7 × 104, p = 0.006) compared to those with true positive RDT (median 2 × 105 copies/µL, IQR 3 × 104 – 5 × 105) and microscopy (median 2 × 105 copies/µL, IQR 6 × 104 – 5 × 105). Conclusions: The study emphasizes that malaria was a frequent cause of febrile illness in hospitalized patients in Zanzibar in the years 2015-2016, particularly among school age children and young adults. We found evidence of autochthonous malaria transmission in Zanzibar. Compared to PCR, both RDT and microscopy had low sensitivity, and false negative results were associated with low parasitaemia. While low parasitaemia identified only by PCR in a semi-immune individual could be coincidental and without clinical relevance, clinicians should be aware of the risk of false negative results on routine tests.publishedVersio

Diagnosis and follow-up of treatment of latent tuberculosis; the utility of the QuantiFERON-TB Gold In-tube assay in outpatients from a tuberculosis low-endemic country

<p>Abstract</p> <p>Background</p> <p>Interferon-gamma (IFN-γ) Release Assays (IGRA) are more specific than the tuberculosis skin test (TST) in the diagnosis of latent tuberculosis (TB) infection (LTBI). We present the performance of the QuantiFERON^®-TB Gold In-tube (QFT-TB) assay as diagnostic test and during follow-up of preventive TB therapy in outpatients from a TB low-endemic country.</p> <p>Methods</p> <p>481 persons with suspected TB infection were tested with QFT-TB. Thoracic X-ray and sputum samples were performed and a questionnaire concerning risk factors for TB was filled. Three months of isoniazid and rifampicin were given to patients with LTBI and QFT-TB tests were performed after three and 15 months.</p> <p>Results</p> <p>The QFT-TB test was positive in 30.8% (148/481) of the total, in 66.9% (111/166) of persons with origin from a TB endemic country, in 71.4% (20/28) previously treated for TB and in 100% (15/15) of those diagnosed with active TB with no inconclusive results. The QFT-TB test was more frequently positive in those with TST ≥ 15 mm (47.5%) compared to TST 11-14 mm (21.3%) and TST 6-10 mm (10.5%), (p < 0.001). Origin from a TB endemic country (OR 6.82, 95% CI 1.73-26.82), recent stay in a TB endemic country (OR 1.32, 95% CI 1.09-1.59), duration of TB exposure (OR 1.59, 95% CI 1.14-2.22) and previous TB disease (OR 11.60, 95% CI 2.02-66.73) were all independently associated with a positive QFT-TB test. After preventive therapy, 35/40 (87.5%) and 22/26 (84.6%) were still QFT-TB positive after three and 15 months, respectively. IFN-γ responses were comparable at start (mean 6.13 IU/ml ± SD 3.99) and after three months (mean 5.65 IU/ml ± SD 3.66) and 15 months (mean 5.65 IU/ml ± SD 4.14), (p > 0.05).</p> <p>Conclusion</p> <p>Only one third of those with suspected TB infection had a positive QFT-TB test. Recent immigration from TB endemic countries and long duration of exposure are risk factors for a positive QFT-TB test and these groups should be targeted through screening. Since most patients remained QFT-TB positive after therapy, the test should not be used to monitor the effect of preventive therapy. Prospective studies are needed in order to determine the usefulness of IGRA tests during therapy.</p

PCR based Malaria diagnostics : – Method development and application

Background Almost half of the world’s population is at risk of malaria. Malaria is a parasitic vector borne disease spread by infected female Anopheles mosquitos. The protozoan parasite belongs to the genus Plasmodium group. In humans five main species are described, P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. The recent years the WHO has reported that over 200 million malaria cases, and 400 000 deaths occur every year. Microscopy and rapid diagnostic tests (RDTs) are used in routine malaria diagnostics, but these methods have their challenges in accurate sensitivity and specificity. Aim To develop a new robust and user-friendly PCR method, highly sensitive and specific, and to assess and apply this method on relevant clinical material. Methods We developed a new single-step genus-specific conventional PCR targeting the cytochrome b gene (cytb) on the mitochondrial genome, and modified a species-specific conventional PCR targeting the chromosomal small subunit ribosomal RNA 18S locus. The methods were assessed and compared with routine microscopy and a gold standard nested 18S PCR using a Norwegian clinical collection (N=135), before applied in a multi-centre hospital-based study in India (N=1412). We also converted the conventional genus-specific cytb PCR to two real-time PCR assays applying SYBR Green and a TaqMan probe. The two assays were assessed using reference material, the Norwegian material (N=113) and a Tanzanian P. falciparum field collection (N= 74), and compared to five other relevant real-time PCR methods. Sequencing of the genus-specific cytb PCR products was also performed. Results Among the Norwegian sample collection (N=135) the novel genus-specific cytb PCR had a 100% sensitivity (28/28), nested 18S PCR 96% (27/28), and microscopy 93% (26/28). The 28 positive cytb PCR products were sequenced revealing six single nucleotide polymorphisms (SNPs) and one insert/deletion allowing for species determination of the 28 sequences. In the Indian multi-centre study, the cytb PCR found a malaria prevalence of 19% (268/1412). Overall, 46% P. falciparum and 38% P. vivax single infections were detected, while P. falciparum and P. vivax double infection was found in 11%, and P. malariae in 5% of the admitted fever patients. Routine microscopy and RDT had a low sensitivity compared to PCR, 29% and 24% respectively. Furthermore, the conventional cytb PCR was successfully converted to real-time PCR. The SYBR assay showed a higher sensitivity and specificity than using a probe. Focusing on detecting low-level parasitaemia, SYBR was also more beneficial to apply than probe due to melting curve analysis (MCA) can reveal unspecific binding or primer-dimers. Compared to the five other relevant real-time PCR methods our cytb SYBR PCR was the most sensitive genus-specific method. Conclusion Choice of amplification target is relevant for achieving high sensitivity in detecting low-level parasitaemia, and it is advantageous to use an intercalating fluorescence dye suitable for MCA. The highly sensitive, specific and user-friendly cytb SYBR PCR can be a useful tool in epidemiological and surveillance research, as well as for clinical malaria diagnostics. The method is genus-specific, which is an advantage in large screening projects. Among ambiguous samples, and in settings where species-specific PCR fails to detect low-level parasitaemia, confirmation and species identification by sequencing of the genus-specific real-time PCR products can be used as a contingency

Detection of remaining Plasmodium DNA and gametocytes during follow up after curative malaria treatment among returned travellers in Norway

Background PCR can be positive weeks after effective malaria treatment, potentially leading to over diagnose of recrudescence and re-infections. The DNA detected by PCR post-treatment might stem from residuals of destroyed asexual parasites, or from live gametocytes. The objective of this clinical observational study was to describe the presence of positive PCR for Plasmodium falciparum and Plasmodium vivax in follow-up samples post-treatment from returned travellers, and the proportion of positive PCR due to gametocytes. Methods Whole blood was collected during hospitalization and outpatient routine follow-up from 13 patients with imported malaria. DNA was extracted applying QIAamp DNA Blood Mini Kit, while mRNA was collected and extracted applying PAXgene Blood RNA Tubes and Kit. All DNA samples (N = 25) were analysed with a genus-specific cytb real-time SYBR PCR, and P. falciparum DNA samples (N = 22) were also analysed with a falciparum-specific varATS real-time TaqMan PCR. All the mRNA samples (N = 18) were analysed with both a genus-specific 18S rRNA RT-PCR and a gametocyte-specific Pfs25 (P. falciparum)/Pvs25 (P. vivax) RT-PCR. Results Latest samples were collected at day 1 (n = 2) and from day 11–54 (n = 11) after treatment. Genus DNA cytb PCR was positive up to 49 days after effective treatment, and 18S rRNA transcripts from active P. falciparum parasites were detectable for at least 11 days. Gametocyte-specific mRNA was detected at latest only two days after treatment. Among six patients with late positive PCR for P. falciparum, four had high parasitaemia at admittance (6–30%), while two had parasitaemia < 2%. Late detection of P. vivax was not found by any of the PCR methods. Conclusions DNA-based PCR can be positive up to at least seven weeks after curative malaria treatment, potentially leading to over-diagnose of recrudescence and re-infections. Based on the observations in this study, it is unclear if the DNA origins from residuals of destroyed parasites or live gametocytes, warranting further investigations

Single amplification PCR targeting mitochondrial genome more sensitive in diagnosing malaria than nested 18S PCR among returned travelers in Bergen, Norway

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Challenges in diagnosing pediatric malaria in Dar es Salaam, Tanzania

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Serological profile among patients with malaria in secondary community hospitals in India - a multicenter cross sectional study

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Malaria prevalence among patients with acute undifferentiated fever in secondary hospitals in India

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