Comparative analysis of four malaria diagnostic tools and implications for malaria treatment in southwestern Nigeria.

OBJECTIVES: One of the problems encountered in malaria control and elimination is inaccurate diagnosis, resulting from the degree of sensitivity of the different malaria diagnostic tools. Even though microscopy remains the gold standard for malaria diagnosis, more sensitive and robust diagnostic tools such as polymerase chain reactions (PCR) are used in research settings to monitor interventions and track sub-microscopic infections due to some of the drawbacks of microscopy. Since diagnosis is a critical determinant for rational malaria treatment, it is imperative that accurate diagnosis must be assured for an effective treatment plan. Therefore, this study compared two routinely used point of care malaria diagnostic tools with two molecular tools and discussed their implication for malaria treatment. DESIGN: In this study, 436 individuals with suspected malaria were sampled and systematically tested using four methods, namely rapid diagnostic test (henceforth referred to as malaria RDT- mRDT), microscopy, nested PCR (nPCR), and quantitative PCR (qPCR). Test sensitivities and specificities were compared, and their level of concordance was determined. RESULTS: With nPCR as the gold standard, a false positivity rate of 42.2%, 8.9%, and 57.8% was obtained for mRDT, microscopy, and qPCR. Similarly, false negativity rates of 12.5%, 62.5%, and 0.8% were obtained for each of the methods mentioned above, respectively. Of all the tools assessed, qPCR gave the highest sensitivity (99.2%) and moderate specificity (42.2%), followed by the mRDT kit used (87.5%). CONCLUSIONS: With the detection of a high false positivity rate based on mRDT and a substantial proportion of sub-microscopic carriers in this study area by nested/quantitative PCR, we recommend that these molecular tools should be in specialized laboratories within the region to (i) track and treat sub-microscopic carriers to prevent their contribution to malaria transmission; (ii) provide reliable epidemiological data using high throughput testing tools for evaluating malaria interventions

Analysis of pfhrp2 genetic diversity in Senegal and implications for use of rapid diagnostic tests

Background: The Senegalese National Malaria Control Programme has recommended use of rapid diagnostic tests (RDTs) that target the histidine-rich protein 2 (HRP2), specific to Plasmodium falciparum, to diagnose malaria cases. The target antigen has been shown to be polymorphic, which may explain the variability in HRP2-based RDT results reported in field studies. The genetic diversity of the pfhrp2 gene has not been investigated in depth in many African countries. The goal of this study is to determine the extent of polymorphism in pfhrp2 among Senegal, Mali and Uganda parasite populations, and discuss the implications of these findings on the utility of RDTs that are based on HRP2 detection. Methods: Sequencing data from the pfhrp2 locus were used to analyze the genetic diversity of this gene among three populations, with different transmission dynamics and malaria parasite ecologies. Nucleotide diversity (π) and non-synonymous nucleotide diversity (πNS) were studied in the pfhrp2 gene from isolates obtained in Senegal. Amino acid repeat length polymorphisms in the PfHRP2 antigen were characterized and parameters of genetic diversity, such as frequency and correlation between repeats in these populations, were assessed. Results: The diversity survey of the pfhrp2 gene identified 29 SNPs as well as insertion and deletion polymorphisms within a 918 bp region. The Senegal pfhrp2 exhibited a substantial level of diversity [π = 0.00559 and πNS = 0.014111 (πS = 0.0291627)], similar to several polymorphic genes, such as msp1, involved in immune responses, and the gene encoding the SURFIN polymorphic antigen, which are surface exposed parasite proteins. Extensive repeat length polymorphisms in PfHRP2, as well as similar patterns in the number, organization and the type of predicted amino acid repeats were observed among the three populations, characterized by an occurrence of Type 2, Type 4 and Type 7 repeats. Conclusions: These results warrant deeper monitoring of the RDT target antigen diversity and emphasize that development of other essential genes as a target for diagnostic tools is critical

Evaluation of the multiplex real time PCR DermaGenius assay for the detection of dermatophytes in hair samples from Senegal

peer reviewe

Molecular identification of Plasmodium species responsible for malaria reveals Plasmodium vivax isolates in Duffy negative individuals from southwestern Nigeria

Abstract Background Malaria in Nigeria is principally due to Plasmodium falciparum and, to a lesser extent to Plasmodium malariae and Plasmodium ovale. Plasmodium vivax is thought to be absent in Nigeria in particular and sub-Saharan Africa in general, due to the near fixation of the Duffy negative gene in this population. Nevertheless, there are frequent reports of P. vivax infection in Duffy negative individuals in the sub-region, including reports from two countries sharing border with Nigeria to the west (Republic of Benin) and east (Cameroon). Additionally, there were two cases of microscopic vivax-like malaria from Nigerian indigenous population. Hence molecular surveillance of the circulating Plasmodium species in two states (Lagos and Edo) of southwestern Nigeria was carried out. Methods A cross-sectional survey between September 2016 and March 2017 was conducted. 436 febrile patients were included for the present work. Venous blood of these patients was subjected to RDT as well as microscopy. Further, parasite DNA was isolated from positive samples and PCR diagnostic was employed followed by direct sequencing of the 18S rRNA of Plasmodium species as well as sequencing of a portion of the promoter region of the Duffy antigen receptor for chemokines. Samples positive for P. vivax were re-amplified several times and finally using the High Fidelity Taq to rule out any bias introduced. Results Of the 256 (58.7%) amplifiable malaria parasite DNA, P. falciparum was, as expected, the major cause of infection, either alone 85.5% (219/256; 97 from Edo and 122 from Lagos), or mixed with P. malariae 6.3% (16/256) or with P. vivax 1.6% (4/256). Only one of the five P. vivax isolates was found to be a single infection. DNA sequencing and subsequent alignment of the 18S rRNA of P. vivax with the reference strains displayed very high similarities (100%). Remarkably, the T-33C was identified in all P. vivax samples, thus confirming that all vivax-infected patients in the current study are Duffy negative. Conclusion The present study gave the first molecular evidence of P. vivax in Nigeria in Duffy negative individuals. Though restricted to two states; Edo in South–South and Lagos in South-west Nigeria, the real burden of this species in Nigeria and sub-Saharan Africa might have been underestimated, hence there is need to put in place a country-wide, as well as a sub-Saharan Africa-wide surveillance and appropriate control measures

A Comparative Study on Phenotypic versus ITS-Based Molecular Identification of Dermatophytes Isolated in Dakar, Senegal

International audienc

Pulmonary Madurella mycetomatis mycetoma secondary to knee eumycetoma, Senegal.

Mycetoma is a neglected tropical disease which is endemic in Senegal. Although this subcutaneous mycosis is most commonly found on the foot, extrapodal localisations have also been found, including on the leg, knee, thigh, hand, and arm. To our knowledge, no case of blood-spread eumycetoma has been reported in Senegal. Here, we report a case of pulmonary mycetoma secondary to a Madurella mycetomatis knee eumycetoma. The patient was a 41-year-old farmer living in Louga, Senegal, where the Sudano-Sahelian climate is characterised by a short and unstable rainy season and a steppe vegetation. He suffered a trauma to the right more than 20 years previously and had received treatment for more than 10 years with traditional medicine. He consulted at Le Dantec University Hospital in Dakar for treatment of a right knee mycetoma which had been diagnosed more than 10 years ago. He had experienced a chronic cough for more than a year; tuberculosis documentation was negative. Grains collected from the knee and the sputum isolated M. mycetomatis, confirmed by the rRNA gene ITS regions nucleotide sequence analysis. An amputation above the knee was performed, and antibacterial and antifungal therapy was started with amoxicillin-clavulanic acid and terbinafine. The patient died within a month of his discharge from hospital

Status of Artemisinin Resistance in Malaria Parasite Plasmodium falciparum from Molecular Analyses of the Kelch13 Gene in Southwestern Nigeria

Evolution and spread of malaria parasite Plasmodium falciparum capable of evading antimalarials are the prime concern to malaria control. The currently effective drug, artemisinin (ART), is under threat due to detection of ART-resistant P. falciparum parasites in the Southeast Asian countries. It has been shown that amino acid (AA) mutations at the P. falciparum Kelch13 (Pfk13) gene provide resistance to ART. Nigeria, a part of the Sub-Saharan Africa, is highly endemic to malaria, contributing quite significantly to malaria, and resistance to chloroquine (CQ) and sulfadoxine-pyrimethamine (SP) combination drugs has already been reported. Since artemisinin combined therapy (ACT) is the first-line drug for treatment of uncomplicated malaria in Nigeria and five amino acid mutations have been validated in the Pfk13 gene alongside with candidate mutations for ART resistance, we performed molecular surveillance for mutations (following PCR and DNA sequence analyses) in this gene from two southwestern states of Nigeria. Statistical analyses of DNA sequences were also performed following different evolutionary models. None of the different validated and candidate AA mutations of Pfk13 gene conferring resistance to ART could be detected in P. falciparum sampled in the two southwestern states of Nigeria. In addition, DNA sequencing and sequence analyses indicated neither evolutionary selection pressure on the Pfk13 gene nor association of mutations in Pfk13 gene with mutations of other three genes conferring resistance to CQ and SP. Therefore, based on the monomorphism at the Pfk13 gene and nonassociation of mutations of this gene with mutations in three other drug-resistant genes in malaria parasite P. falciparum, it can be proposed that malaria public health is not under immediate threat in southwestern Nigeria concerning ART resistance

Quality control of malaria microscopy reveals misdiagnosed non-falciparum species and other microscopically detectable pathogens in Senegal

Abstract Background In developing countries, malaria diagnosis relies on microscopy and rapid diagnostic tests. In Senegal, national malaria control program (NMCP) regularly conducts supervisory visits in health services where malaria microscopy is performed. In this study, expert microscopists assessed the performance of laboratory technicians in malaria microscopy. Methods The present external quality assessment (EQA) was conducted in three different areas of malaria transmission. Participants were laboratory technicians previously trained by NMCP on malaria microscopy. Stored read slides were randomly collected for blinded re-checking by expert microscopists. At the same time a set of 8 slides (3 positive P. falciparum and 5 negative slides) were submitted to participants for proficiency testing. Microscopists performance were evaluated on the basis of the errors rates on slide reading—high false positive (HFP), high false negative (HFN), low false positive (LFP) and low false negative (LFN)—and the calculation of their sensitivities and specificities relative to expert microscopy. Data were entered and analysed using Microsoft Excel software. Results A total of 450 stored slides were collected from 17 laboratories for re-checking. Eight laboratories scored 100% of correct reading. Only one major error was recorded (HFP). Six laboratories recorded LFN results: Borrelia, P. ovale, and low parasite densities (95 and 155 p/μl) were missed. Two P. falciparum slides were misidentified as P. malariae and one P. ovale slide as P. vivax. The overall sensitivities and specificities for all participants against expert microscopists were 97.8 and 98.2% respectively; Sensitivities and specificities of hospital microscopists (96.7 and 98.9%) were statistically similar to those of health centre microscopists (98.5 and 97.8% respectively) (p = 0.3993 and p = 0.9412 respectively). Overall, a very good agreement was noted with kappa value of 0.96 (CI95% 93.4–98.6%) relative to expert microscopy. Proficiency testing showed that among the 17 participants, 11 laboratories scored 100% of correct reading. Three LFN and four LFP results were recorded respectively. The P. falciparum slide with Maurer dots was misidentified as P. ovale in 1 centre and the same slide was misread as P. vivax in another centre; No major error (HFP or HFN) was noted. Conclusion EQA of malaria microscopy showed an overall good performance especially regarding P. falciparum detection. However, efforts need to be made addressing the ability to detect non-falciparum species and others endemic blood pathogens such as Borrelia. The further NMCP training sessions and evaluations should consider those aspects to expect high quality-assured capacity for malaria microscopy