Owusu et al. Malar J (2018) 17:92 https://doi.org/10.1186/s12936-018-2231-7 Malaria Journal RESEARCH Open Access Plasmodium falciparum diagnostic tools in HIV-positive under-5-year-olds in two ART clinics in Ghana: are there missed infections? Ewurama D. A. Owusu1,2*, Samson K. Djonor2, Charles A. Brown2, Martin P. Grobusch1,4,5 and Petra F. Mens1,3 Abstract Background: Plasmodium falciparum, the most dominant species in sub-Saharan Africa, causes the most severe clinical malaria manifestations. In resource-limited Ghana, where malaria and HIV geographically overlap, histidine-rich protein 2 (HRP2)-based rapid diagnostic test (RDT) is a faster, easier and cheaper alternative to clinical gold standard light microscopy. However, mutations in parasite hrp2 gene may result in missed infections, which have severe impli- cations for malaria control. Methods: The performance of a common HRP2-based RDT and expert light microscopy in HIV-positive and HIV-neg- ative children under 5 years old was compared with PCR as laboratory gold standard. Finger-prick capillary blood was tested with First Response® Malaria Ag P. falciparum (HRP2). Giemsa-stained thick and thin blood films were examined with ≥ 200 high power fields and parasites counted per 200 white blood cells. Nested PCR species identification of P. falciparum was performed and resolved on agarose gel. False negatives from RDT were further tested for deleted pfhrp2/3 and flanking genes, using PCR. The study was performed in two anti-retroviral therapy clinics in Accra and Atibie. Results: Out of 401 participants enrolled, 150 were HIV positive and 251 HIV negative. Malaria was more prevalent in children without HIV. Microscopy had a higher sensitivity [100% (99–100)] than RDT [83% (53.5–100)]. Parasites with pfhrp2/3 deletions contributed to missed infections from RDT false negatives. Conclusion: Circulation of malaria parasites with pfrhp2/3 deletions in this population played a role in missed infec- tions with RDT. This ought to be addressed if further strides in malaria control are to be made. Keywords: Malaria, Microscopy, RDT, Diagnostic, Under-fives, HIV, Ghana Background the biomass of Plasmodium and results in an increase HIV and malaria geographically overlap in Ghana [1, 2]. in emergence of anti-malarial resistance [7]. This was HIV co-infection has been shown to increase the vul- based on the assumption that biomass increment will nerability of the population to malaria [3–5]. Out of the increase the rate of mutations, if the probability of de 9248 AIDS deaths reported in Ghana in 2014, 1295 (14%) novo resistance mutations is evenly distributed among were children; and 31.6% of the children who died from all parasites [7]. Hence, drug administration will select AIDS were under 5 years old. This age group is also con- for genetic mutations and spread drug resistance [7]. As sidered a malaria-vulnerable group [6]. A mathemati- such, a substantial contribution to malaria transmission cal model has been used to postulate that HIV increases and drug resistance might originate from this sub-pop- ulation of patients living with HIV. It is imperative that missed malaria infections in this population are minimal, *Correspondence: edampadu@chs.edu.gh 1 Division of Internal Medicine, Department of Infectious Diseases, Centre in order to control malaria transmission and reduce drug of Tropical Medicine and Travel Medicine, Academic Medical Centre, resistance. University of Amsterdam, Amsterdam, The Netherlands Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Owusu et al. Malar J (2018) 17:92 Page 2 of 7 Improved rapid diagnostic tests (RDT), which are participants were recruited from the out-patient depart- faster, cheaper and easier compared to the gold standard ment (OPD) of the main hospital. For this category, only expert microscopy, are complementing malaria control patients who had their HIV-negative status clearly indi- efforts in resource-limited sub-Saharan African countries cated in their folders were included in this study. such as Ghana [8]. In Ghana, most of the rapid diagnos- tic test kits that are available are based on histidine-rich Study design and participant selection protein-2 (HRP2) [9–11], which is an antigen exclu- This was a cross-sectional study, performed from May sive to P. falciparum [12]. However, recent reports have (beginning of the major raining season) to July 2015. shown that hrp-2 deletions in parasite genes (pfhrp2), The study was explained to parents of all children under which previously prevailed only in the South Americas, 5  years old before enrollment. Consecutive patients have now been detected in African countries like Ghana, attending the ART clinics (for HIV positive participants) Democratic Republic of Congo, Eritrea, Uganda and and OPD of the main hospital (for HIV negative partici- Rwanda, regardless of level of transmission [8]. The lack pants) were recruited into the study. Patient information of pfhrp2 expression can prevent the detection of para- such as age, sex, signs and symptoms were recorded. sites by HRP2-based RDTs and give false negative results After malaria screening, participants were classified into [8]. asymptomatic or symptomatic groups. Those who had In spite of the preponderance of HRP2-based RDTs fever (axillary temperature ≥ 37.5 °C) with any of the fol- in Ghana, very limited studies have been conducted on lowing; chills and sweating, general malaise, headache, the circulation of pfhrp2 deletion in parasites in the Gha- vomiting, nausea or diarrhea, were placed in the sympto- naian population. An assessment of diagnostics in the matic category. Bongo District of northern Ghana showed that health workers in the Community-based Health Planning and Study procedure Services (CHPS) had challenges with inaccurate or inva- Malaria screening lid RDT results, which were HRP2-based [13]. However, Finger-prick capillary blood of participants was taken for whilst there was no further investigation into possible cir- three different test procedures. Blood was dropped on culation of parasites with pfhrp2 gene deletions, another First Response® Malaria Ag P. falciparum (HRP2) malaria study found the mutation in Central (40%) and Greater rapid diagnostic test kit and results recorded according Accra (22.4%) regions of Ghana [9]. Missed malaria to the instructions of the manufacturer. This RDT has infections, as a result of inability of RDTs to detect para- passed the lot testing according to WHO-FIND guide- sites, can result in increased morbidity and mortality, lines, which subjects available test kits on the market to especially in children under 5  years old [8]. This could 3–6 rigorous rounds of testing [14]. One of the reasons be more severe in people living with HIV. This study, why it is recommended for this country is because of its therefore, looked at the performance of an HRP2-based stability despite adverse weather conditions [14]. RDT on the Ghanaian market and expert light micros- Thick and thin blood smears were prepared for malaria copy, in under 5 year olds who are living with HIV in two parasite detection and identification. Thin smears were anti-retroviral therapy (ART) centers in Atibie (Eastern fixed in 100% methanol after air-drying. Both were region) and Accra (Greater Accra region), Ghana. stained for 10  min with 10% Giemsa; and examined by two expert microscopists. Enumerating 200 high-pow- Methods ered visual fields, samples were recorded as positive if any Study site and population amount of asexual parasites was observed per 200 white Data was collected at two ART clinics, the Ridge Regional blood cells and negative if none was observed. A third Hospital and Kwahu Government Hospital. The Ridge microscopist resolved discordant results. Participants Regional Hospital is located in the Greater Accra region who tested positive for either RDT or blood smear were whilst the Kwahu Government Hospital is located in the referred to attending doctor for appropriate treatment. Eastern region of Ghana. Malaria prevalence in Greater Three drops of blood were placed on filter paper Accra is relatively lower at 4–10%, whilst that in the East- (Whatman grade 3). These were dried, stored and later ern region is relatively higher at 22% [10]. The vegetation shipped for PCR analysis at the KIT Biomedical Research in Eastern region is forest, whilst that in Greater Accra Institute in Amsterdam, the Netherlands. DNA was is coastal savannah. Malaria transmission is perennial in extracted using the Boom method [15] and nested PCR both study sites. was performed for P. falciparum identification (see [16]). Both clinics provide health services to patients liv- Samples that were RDT negative but PCR positive for P. ing with HIV. They also provide free access to anti-ret- falciparum were further tested for pfhrp2/3 deletion, by roviral drugs, counseling and prophylaxis. HIV negative amplifying the histidine-rich central repeat region using Owusu et al. Malar J (2018) 17:92 Page 3 of 7 primers for the conserved 5′ and 3′ regions of hrp2 and RDT (or microscopy) true negatives, divided by all RDT hrp3 genes. The flanking genes for hrp2 and hrp3 were (or microscopy) negative test results (true negatives plus also amplified. A 25  µl total volume was used for PCR false negatives). reactions, containing 5  µl DNA template, 10× buffer, 25  mM M gCl2, 10  mM each dNTP, 100  mM of each Results primer and 5 units of Taq (Roche diagnostics, Mannheim, General study characteristics Germany). Refer to Additional file 1 for primers used and A total of 150 HIV positive and 251 HIV negative chil- PCR conditions. All PCR products were resolved on 2% dren under 5  years old were recruited into the study. agarose gel electrophoresis. Male:female ratio was 19:131 in the HIV positive group, and 79:172 in the HIV negative group (Table  1). When Statistical analysis RDT was used to test for malaria among both groups, Data was entered in Microsoft Office Excel® 2010 5/150 (3.3%) of HIV positive patients had malaria whilst (Microsoft Corporation, USA), and statistically analysed 44/251 (17.5%) of those without HIV had malaria. Light with XLSTAT statistical ad-in. Diagnostic tools were microscopy and PCR gave similar results in patients liv- compared using McNemar’s test. A p value ≤  0.05 was ing with HIV; 6/150 (4%) participants had malaria. How- considered as statistically significant. Kappa coefficients ever, amongst HIV negative participants, the results of were used to test agreement between the diagnostic tools these two diagnostics did not agree; 50/251 (19.9%) posi- RDT and microscopy. Moderate agreement is indicated tives with light microscopy and 56/251 (22.3%) positives when kappa coefficient is between 0.4 and 0.6, substantial with PCR. Even though 60/150 (40%) of participants agreement when between 0.6 and 0.8 and excellent agree- with HIV and 131/251 (52.2%) without HIV had malaria- ment when 0.8 or more [17]. suggestive symptoms of temperature, headache, vomit- The sensitivity and specificity of RDT and microscopy ing and diarrhoea; malaria parasites were observed only were calculated using PCR as the gold standard. The in 6/150 (4%) with HIV and 56/251 (22.3%) without HIV proportion of RDT and microscopy positives, individu- using PCR (Table 1). ally, divided by the total positives determined by PCR Based on microscopy, the mean parasitaemia of all was recorded as sensitivity. Specificity was calculated patients was 85,050/µl. When the groups were separated as the proportion of individual RDT and microscopy into HIV positive and HIV negative, the mean para- negatives divided by the total negatives in the PCR. The sitaemia were 80,400/µl (19,201–141,559) and 89,700 number of RDT (or microscopy) true positives divided (53,121–126,279), respectively. by the number of  all RDT (or microscopy) positive test results (true positive plus false positives) was recorded Comparison of diagnostic tools and accuracy tests as positive predictive value (PPV); whilst negative pre- There were significant differences between the perfor- dictive value (NPV) was recorded as the number of all mances of RDT, microscopy and PCR (p < 0.05) (Table 2). Table 1 Socio-demographic characteristics and malaria diagnosis of under 5 year olds with HIV and without HIV Category Variable Frequency (%) HIV+ HIV− Age Under 5 150 (100%) 251 (100%) Sex Male 19 (12.7%) 79 (31.5%) Female 131 (87%) 172 (68.5%) RDT diagnostics Negative 145 (96.7%) 207 (82.5%) Positive 5 (3.3%) 44 (17.5%) Light microscopy Negative 144 (96%) 201 (80.1%) Positive 6 (4%) 50 (19.9%) Mean parasite density/µl (CI)a 80,400 (19,201–141,559) 89,700 (53,121–126,279) PCR Negative 144 (96%) 195 (77.7%) Positive 6 (4%) 56 (22.3%) Malaria-like symptoms No 90 (60%) 120 (47.8%) Yes 60 (40%) 131 (52.2%) a Mean of those who tested positive Owusu et al. Malar J (2018) 17:92 Page 4 of 7 Table 2 A comparison of the diagnostic tools RDT, microscopy and PCR; and test of their accuracy RDT/PCR Microscopy/PCR RDT/microscopy Neg 352/339 345/339 352/345 Pos 49/62 56/62 49/56 p value* < 0.05 < 0.05 < 0.05 Kappa co-efficient 0.83 RDT Under 5 years (n = 150) Under 5 years (n = 251) HIV positive (95% CI) HIV negative (95% CI) Sensitivity 83 (53.5–100) 78.6 (67.8–89.3) Specificity 100 (98.9–100) 100 (99.1–100) Positive predictive value 100 (97.8–100) 100 (99.3–100) Negative predictive value 99 (98–100) 94.2 (91–97.4) Microscopy Under 5 years (n = 150) Under 5 years (n = 251) HIV positive (95% CI) HIV negative (95% CI) Sensitivity (CI) 100 (99–100) 89.3 (81.2–97.4) Specificity (CI) 100 (98.5–100) 100 (99.3–100) Positive predictive value 100 (99.2–100) 100 (99–100) Negative predictive value 100 (99.6–100) 97 (94.7–99.4) * p value using McNemar’s test Yet, Kappa coefficient to determine agreement between negatives. However, there was not enough extracted RDT and microscopy showed excellent agreement at DNA in 5 out of the 13 false negatives to perform these 0.83. In general, the sensitivity for expert light micros- tests on. The pfrp2/3 deletion tests on the remaining copy was higher [100% (99–100) and 89.3% (81.2–97.4)] eight showed that the pfhrp2 gene was present in 2 out than RDT [83% (53.5–100) and 78.6% (67.8–89.3)] in of the 8 samples, one of which also had the flanking gene HIV-positive and -negative participants, respectively MAL7P1.228. One other sample had only MAL7P1.228 (Table 2). Specificity of RDT for both HIV-positives and gene (12.5%) (Table 4). None had the pfhrp3 gene nor its -negatives were 100% (98.9–100 and 100% (99.1–100). flanking genes. The positive predictive values (PPV) were 100% (97.8– 100) and 100% (99.3–100) in HIV positive and nega- Discussion tive populations when RDT was used. Microscopy also It has been well established before that malaria and HIV yielded 100% (99.2–100) and 100% (99–100) positive interact manifold with each other [1–4]. The study pop- predictive values. The negative predictive values for RDT ulation for this study had been chosen in the context of were 99% (98–100) and 94.2% (91–97.4) in HIV positive improving care for under-5-year-old HIV patients in and negative individuals, respectively. Negative predic- whom, as with all children in endemic areas, malaria con- tive values were higher with microscopy; 100% (99.6– tinues to constitute a major health threat and frequent 100) among HIV positives and 97% (94.7–99.4) among cause of fever. The problem identified and discussed HIV negatives. here, namely pfhrp2, 3 depletions leading to false nega- All participants living with HIV who tested positive tive malaria RDT results, is not considered specific to with RDT (5/5) were true positives if PCR was used as the HIV-positive population, or the particular young age the gold standard (Table  3). In the HIV-negative group, group. all malaria positives (44/44) obtained with RDT were true This study has demonstrated that the sensitivity and positives. There were no false positives in both HIV nega- specificity of both HRP2-based RDT and expert light tive and HIV positive participants. However, there were microscopy are generally high in rural and urban Ghana false negatives; 1/145 negatives in those with HIV and when tested in children with HIV under 5 years old; and 12/207 in HIV negatives. Minimum parasite densities of microscopy more so than RDT. Missed infections from the false negatives were 17,087 and 11,107 p/µl, respec- microscopy were few, but generally higher in rural Ati- tively, in HIV-positive and HIV-negative groups. bie  (Kwahu). The HRP2-based RDT used in this study Further test was done with PCR to detect pfhrp2 missed 1/6 malaria infections in HIV positive and 12/56 and pfhrp3 and their flanking genes in the RDT false in HIV negative participants. The parasite densities of Owusu et al. Malar J (2018) 17:92 Page 5 of 7 Table 3 The differences in parasite detection amongst the two methods, using PCR as the gold standard Variable HIV+ Minimum parasite density/ HIV− Minimum µl parasite density/µl RDTa True positive 5 19,608 44 53,846 False positive 0 – 0 – Total test positive 5 19,608 44 53,846 False negative 1 17,087 12 11,107 True negative 144 – 195 – Total test negative 145 207 Microscopy True positive 6 19,201 50 53,121 False positive 0 – 0 – Total test positive 6 19,201 50 53,121 False negative 0 – 6 21 True negative 144 – 195 – Total test negative 144 201 PCR Gold standard 6 19,608 56 53,846 a Parasite count for RDT was done with real time PCR Table 4 Pattern of deletion of pfhrp2, pfhrp3 and their flanking genes in RDT false negatives Pfhrp2 exon 2, Pfhrp2 exon 1–2, PFD7_0831900 PFD7_0831700 Pfhrp3 exon 1–2, PF3D7_1372400 PF3D7_1372100 Study Frequency PF3D7_0831800 PFD7_0831800 (MAL7P1.230) (MAL7P1.228) PF3D7_1372200 (MAL13P1.475) (MAL13P1.485) population (%) + + + − − − − HIV negative 1/8 (12.5) + + − − − − − HIV negative 1/8 (12.5) − − + − − − − HIV negative 1/8 (12.5) − − − − − − − Both HIV nega- 5/8 (62.5) tive and positive + + + + + + + Positive – control − − − − − − − Negative – control the missed infections were high (minimum of 11,107 p/ Cape-Coast [9]; whereas in other West African coun- µl); therefore, low parasitaemia could not have been the tries, several studies have found them to abound [8, reason for lack of detection with RDT. Further PCR test 18, 19]. In this study, they were found to circulate in of these false negatives for pfhrp2, pfhrp3 and their flank- both the rural highland and the urban lowland. The ing genes indicated the presence of gene deletions in circulation of these mutants in the population has dire circulation. implications for malaria control in Ghana. Among the Missed infections from microscopy were fewer than malaria-vulnerable population of people living with those from RDT. The false negatives from micros- HIV, the consequence of missed infections may be copy were higher in rural Atibie than in urban Accra. more serious; and even exceedingly so when they are This might be attributed to the subjectivity of expert under 5 years old. As has been established in the litera- microscopy to individual bias. However, state-of-the- ture, malaria morbidity and mortality is worse in chil- art RDTs should be able to rectify that; but false nega- dren under 5 years old [8, 20–22] and in people living tives were observed in the HRP2-based RDT used. with HIV [3, 23]; a combination of these two factors The circulation of pfhrp2 deleted malaria parasites is even more severe [3, 4]. It is, therefore, imperative has been established as one of the possible reasons for that alternative RDTs are used in Ghana, a good option false negatives in RDTs in malaria-endemic areas. One will be ones which detect non-HRP2 antigens. How- of the very few prior studies done in Ghana has shown ever, those that are currently WHO pre-qualified or the deletions to be present in parasites in Accra and meet the WHO recommended procurement criteria Owusu et al. Malar J (2018) 17:92 Page 6 of 7 are insufficient [8]. The development of RDTs that malaria vulnerable sub-population and may contribute detect a combination of antigens might also be benefi- to adaptation of improved control measures. cial, so that mutant parasites with deletions will not be missed. Conclusion The contribution of Plasmodium parasitaemia in Malaria efforts in Ghana are commendable, yet people living with HIV to anti-malarial drug emer- improved focus on malaria vulnerable populations gence has been shown in other studies [7]. Perhaps low such as children under 5  years old living with HIV is malaria prevalence, as found in this HIV population necessary. Current diagnostic tools used, HRP2-based under 5 years of age, might not influence parasite bio- RDT and expert microscopy, have high sensitivities and mass and contribute to de novo resistance mutations. specificities. Nonetheless, missed infections due to the Yet, rather than use parasite count of peripheral blood presence of parasites with deleted pfhrp2 genes need to as an indication of parasite biomass since it does not be addressed if further strides towards malaria elimina- take into consideration sequestered parasite load, there tion are to be made. have been suggestions to use plasma Pfhrp2 concen- trations as estimates [24]. However, in the light of the Additional file circulation of Pfhrp2 deletions in this study and others, this might not be an accurate estimation after all. Additional file 1. PCR reaction conditions and primer sequences. The In a study in other parts of Ghana by Adu-Gyasi et al. conditions and primer sequences for amplification of pfhrp2, pfhrp3 and their flanking genes were adapted from Abdallah et al. [30]. [25], malaria prevalence in HIV positive patients was lower than the average national malaria prevalence. This is similar to the present study results; and may be Authors’ contributions attributed to co-trimoxazole (CTX) prophylaxis (given EDAO, MPG and PM conceived and designed this study. Data acquisition, analysis and interpretation was done by EDAO, SKD, CB, MPG and PM. EDAO with anti-retroviral therapy as recommended by the wrote the first draft of the article, which was critically revised by MPG, PM and Ghana National Aids Control Programme) [26]. All the CB for important intellectual content. All authors read and approved the final under-5-year-olds living with HIV in this study were manuscript. on prophylactic CTX. This has been shown to reduce Author details opportunistic infections and protozoan infections like 1 Division of Internal Medicine, Department of Infectious Diseases, Centre malaria [27–29]. of Tropical Medicine and Travel Medicine, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands. 2 Department of Medical Even though 40% of the children living with HIV Laboratory Sciences, School of Biomedical and Allied Health Sciences, College in this study had symptoms that were indicative of of Health Sciences, University of Ghana, P.O. Box 0s123, Osu, Accra, Ghana. 3 malaria, such as fever, chills and sweating, headache, Division of Laboratory Specialisms, Department of Medical Microbiology, Clinical Parasitology, Academic Medical Centre, University of Amsterdam, general malaise, vomiting, diarrhea or nausea, PCR Amsterdam, The Netherlands. 4 Centre de Recherches Médicales de Lam- showed that only 4% of that category actually had baréné (CERMEL), Hôpital Albert Schweitzer, Lambaréné, Gabon. 5 Institute malaria. In a similar study in people living with HIV in of Tropical Medicine, University of Tübingen, Tübingen, Germany. Brong-Ahafo and Ashanti regions in Ghana, 38.1% of Acknowledgements study participants had malaria symptoms yet only 4.4% The incalculable help of Sandra Menting, Johanna Roth and Nienke Verhaar were laboratory confirmed [25]. This suggests that the in the molecular analysis of hrp2/3 deletion at the Department of Medical Microbiology, Academic Medical Centre, the Netherlands, are very much adoption of the WHO recommendation for laboratory appreciated. diagnosis of malaria instead of the erstwhile clinical diagnosis by Ghana is a step in the right direction. It Competing interestsThe authors declare that they have no competing interests. will reduce prescription of anti-malarials to only when it is necessary. Availability of data and materials Ideally, subject to financing, an expansion of this The datasets generated or analysed during this study are included in this published article (and its additional files). Further data are available from the study to other regions in Ghana should reveal the pat- corresponding author upon reasonable request. tern of distribution of the pfhrp2 mutants. Nonethe- less, this is one of the first studies in Ghana that has Consent for publicationNot applicable. looked at the performance of popular diagnostic tools in a malaria vulnerable population with the added vul- Ethics approval and consent to participate nerability of HIV. It has also detected the circulation of This study was approved by the Ghana Health Service (GHS-ERC: 02/03/14). Before enrollment into study, parents of each under 5 year old gave their parasites with pfhrp2 deletions in this malaria-vulner- written informed consent. The directors of the National AIDS Control Program able population in the rural highland and urban low- (NACP) and the Medical Directors of Ridge Regional Hospital (Accra) and land. This has shed light on the malaria situation of the Kwahu Government Hospital (Atibie) also gave their consent to this study. Owusu et al. Malar J (2018) 17:92 Page 7 of 7 Funding 16. 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Target product profile: point-of-care malaria infection detec- tion test for rapid detection of low-density, subclinical malaria infections. 2014. https ://www.findd x.org/wp-conten t/uploa ds/2016/02/DIAME TER-poc-malar ia-idt-TPP-2014.pdf. Accessed 13 Apr 2017. 13. Agandaa SA, Kweku M, Agboli E, Takase M, Takramah W, Tarkang E, et al. Implementation and challenges of test, treat and track (T3) strategy for Submit your next manuscript to BioMed Central malaria case management in children under five years in the Bongo District, Ghana. Clin Res Trials. 2016;2:235–41. and we will help you at every step: 14. WHO/FIND/CDC. Malaria Rapid Diagnostic Test Performance. Results of WHO product testing of malaria RDTs: round 6 (2014–2015). 2015. p. 154. • We accept pre-submission inquiries 15. Boom R, Sol C, Salimans M, Jansen C, Wertheim-van Dillen P, van der • Our selector tool helps you to find the most relevant journal Noordaa J. Rapid and simple method for purification of nucleic acids. 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