Evaluation of the Multiplex Real-Time PCR DermaGenius® Assay for the Detection of Dermatophytes in Hair Samples from Senegal.

For the successful treatment of dermatophytoses, especially tinea capitis, there is a need for accurate and rapid diagnostic methods. A lot of recent literature has focused on the detection of dermatophytes directly on sample material such as nails, hair and skin scrapings. Molecular tools offer the ability to rapidly diagnose dermatophytosis within 48 h. This study aimed to compare the results of a commercial real-time PCR (real-time PCR) assay DermaGenius®(DG) 2.0 complete multiplex kit with those of conventional diagnostic methods (direct microscopy and culture). A total of 129 hair samples were collected in Dakar (Senegal) from patients suspected of dermatophytosis. DG was applied for the molecular detection of Candida albicans, Trichophyton rubrum/soudanense, T. interdigitale, T. tonsurans, T. mentagrophytes, T. violaceum, Microsporum canis, M. audouinii, Epidermophyton floccosum, T. benhamiae and T. verrucosum. Dermatophytes species and C. albicans were differentiated by melting curve analysis. The sensitivity and specificity of the PCR assay were 89.3% and 75.3%, respectively. DG PCR was significantly more sensitive than culture (p < 0.001). DG PCR is fast and robust to contamination. In this paper, the main questions discussed were the replacement of culture by a broad-spectrum fungal real-time PCR and the implementation of DG PCR into a routine laboratory in Senegal.

Epidemiological and Mycological Aspects of Onychomycosis in Dakar (Senegal).

Onychomycosis is a fungal nails infection often caused by yeasts, dermatophytes and molds. It is an important public health concern due to its high prevalence, the problem of diagnostics, and the poor response to treatments. The objective of this study was to evaluate the epidemiological and microbiological profile of onychomycosis diagnosed at the Laboratory of Parasitology-Mycology of the National University Hospital of Fann in Dakar, Senegal, from 2012 to 2016. A retrospective and descriptive study was performed from January 2012 to December 2016 in a patient attending the laboratory of Parasitology-Mycology at the Fann teaching hospital. Socio-demographic, clinical and biological data were collected from the bench registers. Samples from the lesions were tested using direct microscopy and cultured on a Sabouraud-Chloramphenicol and Sabouraud-Chloramphenicol-Actidione medium. A descriptive analysis was done using Stata IC 12 software. The significance level of different tests was set at 5% two-side. A total of 469 patients were included in this study. The mean age of the study population was 33.2 ± 15.2 years, and the sex ratio was 0.52. The prevalence of onychomycosis was 48.4% (227/469). The main clinical presentations were disto-lateral subungual onychomycosis (37.9%) and onyxis (46.5%). Identified fungal species were Candida albicans (42.7%), Candida spp (39.5%), Trichophyton soudanense (10.1%), Fusarium spp (5.3%), and Candida tropicalis (2.6%). Candida albicans was more frequent in subjects over 15 years of age (43.6%) and women (45%). However, Trichophyton soudanense was higher in patients under 15 years old (17.4%) as well as in male subjects (18.8%). In conclusion, onychomycosis is a common cause of consultation in health facilities. Candida albicans and Trichophyton Soudanense are the main fungal species causing onychomycosis. A better understanding of the epidemiology of onychomycosis as well as the spectrum of the pathogen could contribute to improve the management of the infection.

A Study of Trichomonas vaginalis Infection and Correlates in Women with Vaginal Discharge Referred at Fann Teaching Hospital in Senegal.

INTRODUCTION: Trichomoniasis is nowadays the most prevalent non-viral sexually transmitted infection in the world. In Senegal, the epidemiology of trichomoniasis is not well known. The current study aimed at assessing the prevalence and factors associated with T. vaginalis infection among women with vaginal discharge. METHODS: A retrospective analysis of laboratory records from patients referred at the Fann Teaching Hospital in Dakar, Senegal, for vaginal discharge was carried out. The study covered the period from 2006 to 2011. For each participating woman, a vaginal swab was collected and a wet mount smear performed immediately. Optic microscopic examination with 40x magnification was done to detect T. vaginalis and assess biological modifications such as presence of epithelial cells, white blood cells, and red blood cells. A gram stained smear was also performed and examined under oil immersion (100x magnification) to assess the vaginal flora. RESULTS: Overall, 3893 women were enrolled with a mean age at 31.2 ± 10 years. The prevalence of Trichomoniasis represented 4.8%, 95%CI(3.1-5.7) and it was lower among women less than 30 years (4.1%), while divorced women more likely to be infected compared to married and single women (aOR:2.1, 95%CI (1.2-3.7)). Trichomoniasis was associated with abnormal vaginal flora such as type III (aOR:2.6, 95%CI(1.5-4.4)) and type IV (aOR:3.3, 95%CI(2.1-5.3)). In addition, patients with erythrocytes excretion were more likely to be infected by T. vaginalis (aOR:2.8, 95%CI(1.9-3.9). CONCLUSION: Trichomonas vaginalis remains prevalent among sexually active women. Strategies aiming at improving disease awareness in these high-risk groups are needed to improve trichomoniasis prevention but extensive epidemiological data are still needed for a better understanding of the disease transmission dynamic.

Emergence of uncommon HIV-1 non-B subtypes and circulating recombinant forms and trends in transmission of antiretroviral drug resistance in patients with primary infection during the 2013-2015 period in Marseille, Southeastern France.

Primary HIV-1 infections (PHI) with non-B subtypes are increasing in developed countries while transmission of HIV-1 harboring antiretroviral resistance-associated mutations (RAMs) remains a concern. This study assessed non-B HIV-1 subtypes and RAMs prevalence among patients with PHI in university hospitals of Marseille, Southeastern France, in 2005-2015 (11 years). HIV-1 sequences were obtained by in-house protocols from 115 patients with PHI, including 38 for the 2013-2015 period. On the basis of the phylogenetic analysis of the reverse transcriptase region, non-B subtypes were identified in 31% of these patients. They included 3 different subtypes (3A, 1C, 4F), 23 circulating recombinant forms (CRFs) (CRF02_AG, best BLAST hits being CRF 36_cpx and CRF30 in 7 and 1 cases, respectively), and 5 unclassified sequences (U). Non-B subtypes proportion increased significantly, particularly in 2011-2013 vs in 2005-2010 (P = .03). CRF02_AG viruses largely predominated in 2005-2013 whereas atypical strains more difficult to classify and undetermined recombinants emerged recently (2014-2015). The prevalence of protease, nucleos(t)ide reverse transcriptase, and first-generation nonnucleoside reverse transcriptase inhibitors-associated RAMs were 1.7% (World Health Organization [WHO] list, 2009/2.6% International AIDS Society [IAS] list, 2017), 5.2%/4.3%, and 5.2%/5.2%, respectively. Etravirine/rilpivirine-associated RAM (IAS) prevalence was 4.3%. Men who have sex with men (MSM) were more frequently infected with drug-resistant viruses than other patients (26% vs 7%; P = .011). The recent increase of these rare HIV-1 strains and the spread of drug-resistant HIV-1 among MSM in Southeastern France might be considered when implementing prevention strategies and starting therapies.

Comparison of Para-Selles Bailenger/Kop-Color Fumouze, Para-Selles-Iodésine/Kop-Color II Fumouze diagnostic kits with conventional microscopic methods in identifying intestinal parasitic diseases in Senegal.

In the context of controlling intestinal parasites, accurate diagnosis is essential. Our objective was to evaluate the performance of new diagnostic kits compared to conventional microscopic methods in identifying intestinal parasites. Faeces collected in rural area in Senegal were subjected to several detection techniques. Thus, the sensitivity, specificity, positive and negative predictive values of new diagnostic techniques were compared to conventional merthiolate-iodine-formalin, conventional Bailenger and modified Ritchie. Furthermore, the kappa coefficient was calculated to evaluate the correlation between the new kit and those of modified Ritchie. Out of the 117 patients examined, 102 presented with a parasite, or prevalence of 87.1%. The Fumouze techniques proved to be as effective as the conventional methods in detecting flagellates and helminths with sensitivities ranging from 97 to 100%. However, conventional techniques were slightly more sensitive in identifying Endolimax nana and Blastocystis hominis. The correlation was nearly perfect (k = 0.83 and 1), respectively between Bailenger Fumouze, Iodesine Fumouze and modified Ritchie in identifying helminths while it was just acceptable (k = 0.27 and 0.28) in identifying B. hominis. The modified Ritchie technique routinely used in our laboratory remains a good diagnostic tool. However, the use of kit techniques was interesting when reading the pellet after concentration and the Colour KOP staining was a considerable contribution to the diagnosis of the vegetative forms. Therefore, it would be interesting to determine the cost of a stool test using Fumouze kit techniques to provide the most cost effective way.

Performance of Real-Time Polymerase Chain Reaction Assays for the Detection of 20 Gastrointestinal Parasites in Clinical Samples from Senegal.

Gastrointestinal parasite infections represent one of the biggest public health problems in the world. Therefore, appropriate innovative tools are needed for assessing interventions to control these infections. This study aims to compare the performance of real-time polymerase chain reaction (PCR) assays to microscopic examination for detection of intestinal parasites. A direct microscopic examination and stool concentration was performed on 98 stool samples from patients attending Senegalese hospitals. Negative microscopic control samples were also collected in Nice and Marseille (France). Species-specific primers/probes were used to detect 20 common gastrointestinal protozoans and helminths. Positive frequency and the sensitivity of each real-time PCR assay were compared with conventional microscopic examination. Real-time PCR was positive in 72 of 98 samples (73.5%), whereas microscopic examination was positive in 37 (37.7%) samples (P < 0.001). The real-time PCR assays were more sensitive than microscopy, with 57.4% (31/54) versus 18.5% (10/54), respectively, in the detection of parasites in asymptomatic patients (P < 0.05). In terms of polyparasitism, there were more coinfections detected by real-time PCR assays compared with microscopic methods (25.5% versus 3.06%). In comparison to parasite prevalence on individual samples, the results showed a perfect agreement (100%) between the two techniques for seven species, whereas discrepancies were observed for the others (agreement percentage varying from 64.2% to 98.9%). Real-time PCR appeared to be superior to microscopic examination for the detection of parasites in stool samples. This assay will be useful in diagnostic laboratories and in the field for evaluating the efficacy of mass drug administration programs.

Sero-epidemiological evaluation of Plasmodium falciparum malaria in Senegal.

BACKGROUND: In Senegal, a significant decrease of malaria transmission intensity has been noted the last years. Parasitaemia has become lower and, therefore, more difficult to detect by microscopy. In the context of submicroscopic parasitaemia, it has become relevant to rely on relevant malaria surveillance tools to better document malaria epidemiology in such settings. Serological markers have been proposed as an essential tool for malaria surveillance. This study aimed to evaluate the sero-epidemiological situation of Plasmodium falciparum malaria in two sentinel sites in Senegal. METHODS: Cross-sectional surveys were carried out in Velingara (south Senegal) and Keur Soce (central Senegal) between September and October 2010. Children under 10 years old, living in these areas, were enrolled using two-level, random sampling methods. P. falciparum infection was diagnosed using microscopy. P. falciparum antibodies against circumsporozoite protein (CSP), apical membrane protein (AMA1) and merozoite surface protein 1_42 (MSP1_42) were measured by ELISA method. A stepwise logistic regression analysis was done to assess factors associated with P. falciparum antibodies carriage. RESULTS: A total of 1,865 children under 10 years old were enrolled. The overall falciparum malaria prevalence was 4.99% with high prevalence in Velingara of 10.03% compared to Keur Soce of 0.3%. Symptomatic malaria cases (fever associated with parasitaemia) represented 17.37%. Seroprevalence of anti-AMA1, anti-MSP1_42 and anti-CSP antibody was 38.12, 41.55 and 40.38%, respectively. The seroprevalence was more important in Velingara and increased with age, active malaria infection and area of residence. CONCLUSION: The use of serological markers can contribute to improved malaria surveillance in areas with declining malaria transmission. This study provided useful baseline information about the sero-epidemiological situation of malaria in Senegal and can contribute to the identification of malaria hot spots in order to concentrate intervention efforts. TRIAL REGISTRATION NUMBER: PACTR201305000551876 ( http://www.pactr.org ).

Usefulness of MALDI-TOF Mass Spectrometry for Routine Identification of Candida Species in a Resource-Poor Setting.

BACKGROUND: Identification of fungal clinical isolates is essential for therapeutic management. In resource-limited settings, identification mostly relies on biochemical tests whose sensitivity and specificity are known to be insufficient for identification of closely related or newly described species. MALDI-TOF has been shown in favored countries to be a reliable and powerful tool for microorganism identification, including yeasts. The aim of this study was to compare MALDI-TOF with routine identification procedures in a resource-poor context. METHODS: A total of 734 clinical specimens (502 vaginal swabs, 147 oral swabs, 61 bronchoalveolar lavage fluids and 24 stool samples) have been tested in the mycology unit of Fann Hospital, Dakar, Senegal. Strains isolated from culture were identified by both conventional phenotypic methods (germ tube formation and biochemical panels) and MALDI-TOF Saramis/VITEK MS, bioMérieux, France. In addition to comparing the final identification, we determined the time of obtaining the results and the cost for both approaches. RESULTS: Overall, 218 (29.7 %) samples were positive for Candida. MALDI-TOF MS enabled the identification of 214 of the 218 strains isolated (98.1 %) at species level. Phenotypic approach yielded identification for 208 strains (95.4 %). Congruence between the tests was observed for 203 isolates. A discrepancy was observed for one isolate identified as Candida krusei with the phenotypic approach and Candida tropicalis with the MALDI-TOF. In addition, ten isolates identified at genus level by phenotypic methods were identified as C. glabrata (n = 8), C. tropicalis (n = 1) and C. parapsilosis (n = 1) by MALDI-TOF. The turnaround time for identification was <1 h using the MALDI-TOF compared to our routine procedures (48 h). The overall cost (reagents + expendables) per isolate was at 1.35 for the MALDI-TOF MS. CONCLUSION: MALDI-TOF clearly outperformed the diagnosis capacities of phenotypic methods by reducing the delay of results and giving accurate identification at species level. Moreover, this approach appears to be cost-effective and should be implemented especially in resource-poor context.

Acute kidney injury associated with Plasmodium malariae infection.

According to current estimates, Plasmodium malariae is not very common in Senegal, as more than 98% of malaria cases are suspected to be due to Plasmodium falciparum. However, it is possible that other malarial species are being under-reported or misdiagnosed. This is a report of a case of P. malariae in a 30-year-old man previously hospitalized with acute kidney injury after treatment with quinine and re-hospitalized three months later. He was diagnosed with renal cortical necrosis post malaria treatment. Plasmodium malariae was identified with light microscope and confirmed using species-specific small-subunit rRNA (ssrRNA) amplification.The patient was treated for malaria with intravenous quinine for seven days, followed by three days of oral treatment; the bacterial infection was treated using ceftriaxone during the first hospitalization and ciprofloxacin associated with ceftriaxone the second time. He also had four rounds of dialysis after which he partially recovered the renal function. Given the complications that can be caused by P. malariae infection, it should be systematically looked for, even if the predominant species is P. falciparum in Senegal.

Cryptococcal meningitis in Senegal: epidemiology, laboratory findings, therapeutic and outcome of cases diagnosed from 2004 to 2011.

BACKGROUND: Cryptococcal meningitis is one of the most important opportunistic infection and a major contributor to early mortality. In sub-Saharan Africa, particularly in Senegal, prevalence of cryptococcal meningitis remains high. This study aimed to describe the epidemiology, laboratory profile, therapeutic and outcome of cases diagnosed in Dakar. METHODS: We analyzed the cryptococcosis cases diagnosed at the department of parasitology-mycology in Fann Teaching Hospital in Dakar from 2004 to 2011. The diagnosis was confirmed by culture on Sabouraud's dextrose agar and/or by India ink preparation and/or by cryptococcal antigen detection. The diagnosis methods were assessed by using culture as reference. RESULTS: A total of 106 cases of cryptococcal meningitis were diagnosed. The prevalence of cryptococcal meningitis was 7.8 %. The mean age of the patients was 40.17 ± 9.89 years. There were slightly more male (53.8 %) than female (46.2 %) patients; 89.6 % were found to be infected with HIV, and the median CD4+ count was 27/mm(3). Approximately 79.5 % of the patients had <100 CD4+ lymphocytes/mm(3). India ink staining presented sensitivity at 94.11 % and specificity at 100 %. Sensitivity and specificity of cryptococcal antigen detection in cerebrospinal fluid were, respectively, 96.96 and 15.78 %. The most frequently used antifungal drug was fluconazole (86.7 %), and the mortality rate was 62.2 % (66 deaths). CONCLUSION: Early diagnosis is essential to control cryptococcosis, and countries should prioritize widespread and reliable access to rapid diagnostic cryptococcus antigen assays. But it is important to make available conventional methods (India ink and culture) in the maximum of laboratory in regional health facilities.

Evolution of the pfcrt T76 and pfmdr1 Y86 markers and chloroquine susceptibility 8 years after cessation of chloroquine use in Pikine, Senegal.

The goal of the present study was to assess the evolution of the in vitro chloroquine resistance and also the prevalence of pfcrt T76 and pfmdr1 Y86 mutations in Pikine from 2000 while chloroquine (CQ) was the first-line treatment of malaria to 2009 when artemisinin-based combination therapies (ACTs) are in use. We genotyped pfcrt K76T and pfmdr1 N86Y polymorphisms by PCR-RFLP and assessed in vitro CQ susceptibility by double-site enzyme-linked pLDH immunodetection (DELI) assay in Plasmodium falciparum isolates collected in Pikine, Senegal. The proportions of the pfcrt T76 allele in the light of the three different treatment policies were 72.4 % before CQ withdrawal (2000 to 2003), 47.2% while amodiaquine plus Fansidar was the first-line treatment (2004 to 2005), and 59.5 % since the ACT use was implemented (2006 to 2009). The prevalence of pfcrt T76 decreased significantly after CQ was stopped [X (2) = 6.54, P = 0.01 (2000-2003 versus 2004-2005)] and then slightly since ACTs have been implemented [X(2) = 1.12, P = 0.28 (2000-2003 versus 2006-2009)]. There were no significant differences on the prevalence of pfmdr1 Y86 throughout the three treatment policies. The DELI assay was carried out episodically in 2000 (n = 36), 2001 (n = 47), and 2009 (n = 37). The mean IC(50)s of the isolates to CQ in 2000 versus 2009 and 2001 versus 2009 are significantly different (P < 0.05). The Fisher exact test found a significant association between the presence of the pfcrt T76 mutant allele and in vitro resistance in 2000/2001 (P = 0.023), while in 2009 there were no association between both variables (P = 0.274). Mutant pfcrt T76 and pfmdr1 Y86 alleles and in vitro CQ-resistant strains are still circulating in Pikine. The official discontinuation of CQ use is not completely followed by its total withdrawal from private drug sellers, and the molecule still exerts pressure on local P. falciparum populations.

Ultrastructure of the spermatozoon of Taeniarhynchus saginatus (syn. Taenia saginata) (Goeze, 1782) Weinland, 1858 (Cestoda, Taeniidae) an intestinal parasite of human.

The mature Taeniarhynchus saginatus spermatozoon exhibits an apical cone of electron-dense material and one helicoidal crest-like body roughly 50 nm thick. The axoneme is of the 9 + "1" Trepaxonemata pattern. It is surrounded by a periaxonemal sheath of electron-dense material. The cytoplasm is electron lucent and divided into compartments by intracytoplasmic walls of electron-dense material in regions III and IV. The nucleus is an electron-dense cord 60-90 nm thick coiled in a spiral around the axoneme. It reaches the posterior extremity of the gamete where the axoneme is disorganized and is accompanied on all its posterior length by the nucleus. To our knowledge, such a posterior extremity has never been described before in a cyclophyllidean cestode.

In vivo and in vitro analysis of chloroquine resistance in Plasmodium falciparum isolates from Senegal.

To determine the predictive value of chloroquine (CQ) resistance markers in Senegal, Plasmodium falciparum DNA polymorphisms in pfmdr1and pfcrt were examined in relation to clinical outcome. Despite CQ treatment, 17% of patients had parasitemia after 28 days. Examination of molecular markers of CQ resistance revealed that 64% of all isolates had the T76 resistant allele at the pfcrt locus, while 30% carried the Y86 resistant allele at the pfmdr1 locus. The pfcrt T76 allele was present not only in all in vivo resistant isolates, 89% of in vitro resistant isolates, but also in 35% of in vitro sensitive isolates. The pfmdr1 N86Y polymorphism did not correlate with in vitro or in vivo CQ resistance. Our data suggest that the pfcrt T76 allele alone is required but not a sufficient predictor for in vivo CQ resistance.

Analysis of the genetic diversity of the Plasmodium falciparum multidrug resistance gene 5' upstream region.

Recent findings indicating a low level of polymorphism in the Plasmodium falciparum genome have led to the hypothesis that existent polymorphisms are likely to have functional significance. We tested this hypothesis by developing a map of the polymorphism in the P. falciparum multidrug resistance 1 (pfmdr1) gene 5' upstream region and assaying its correlation with drug resistance in a sample of field isolates from Dakar, Senegal. A comparison of six geographically diverse laboratory strains showed that the 1.94-kb 5'-untranslated region is highly monomorphic, with a total of four unique single nucleotide polymorphisms (SNPs) being identified. All of the mutations were localized to a 462-basepair region proximal to the transcription start point. Analysis of this region in field isolates shows the prevalence of one SNP throughout the entire population of parasites, irrespective of drug resistance status. The SNP frequency of the pfmdr1 upstream region is lower than that found in the noncoding region of other genes.

In vitro chloroquine susceptibility and PCR analysis of pfcrt and pfmdr1 polymorphisms in Plasmodium falciparum isolates from Senegal.

Chloroquine resistance has been linked to mutations in the pfcrt and pfmdr1 genes of Plasmodium falciparum. To estimate the prevalence of the pfcrt K76T, pfmdr1 N86Y, and pfmdr1 D1246Y polymorphisms, isolates of P. falciparum from Senegal, West Africa, were analyzed, and the results were compared to in vitro chloroquine susceptibility. By the in vitro DELI test, 31% of these samples were resistant to chloroquine. Polymerase chain reaction-based assays and confirmatory sequencing demonstrated the pfcrt T76, pfmdr1 Y86, and pfmdr1 Y1246 alleles in 79%, 31%, and 2% of the isolates, respectively. All three mutant alleles were present in both in vitro susceptible and resistant isolates. On the basis of these findings, it appears that these molecular markers are not consistently predictive of in vitro chloroquine resistance in Senegal.