Alexandra Kerleguer

Field trial evaluation of the performances of point-of-care tests for screening G6PD deficiency in Cambodia.

Background User-friendly, accurate, point-of-care rapid tests to detect glucose-6-phosphate dehydrogenase deficiency (G6PDd) are urgently needed at peripheral level to safely recommend primaquine for malaria elimination. Methods The CareStart G6PD RDT (AccessBio, New Jersey, USA), a novel rapid diagnostic test and the most commonly used test, the fluorescent spot test (FST) were assessed against the quantitatively measured G6PD enzyme activity for detecting G6PDd. Subjects were healthy males and non-pregnant females aged 18 years or older residing in six villages in Pailin Province, western Cambodia. Findings Of the 938 subjects recruited, 74 (7.9%) were severe and moderately severe G6PD deficient (enzyme activity <30%), mostly in male population; population median G6PD activity was 12.0 UI/g Hb. The performances of the CareStart G6PD RDT and the FST, according to different cut-off values used to define G6PDd were very similar. For the detection of severe and moderately severe G6PDd (enzyme activity <30%, <3.6 UI/g Hb) in males and females, sensitivity and negative (normal status) predictive value were 100% for both point-of-care tools. When the G6PDd cut-off value increased (from <40% to <60%), the sensitivity for both PoCs decreased: 93.3% to 71.7% (CareStart G6PD RDT, p = 10−6) and 95.5% to 73.2% (FST, p = 10−6) while the specificity for both PoCs remained similar: 97.4% to 98.3% (CareStart G6PD RDT, p = 0.23) and 98.7% to 99.6% (FST, p = 0.06). The cut-off values for classifying individuals as normal were 4.0 UI/g Hb and 4.3 UI/g Hb for the CareStart G6PD RDT and the FST, respectively. Conclusions The CareStart G6PD RDT reliably detected moderate and severe G6PD deficient individuals (enzyme activity <30%), suggesting that this novel point-of-care is a promising tool for tailoring appropriate primaquine treatment for malaria elimination by excluding individuals with severe G6PDd for primaquine treatment.

Tolerability and safety of weekly primaquine against relapse of Plasmodium vivax in Cambodians with glucose-6-phosphate dehydrogenase deficiency

BackgroundPrimaquine is used to prevent Plasmodium vivax relapse; however, it is not implemented in many malaria-endemic countries, including Cambodia, for fear of precipitating primaquine-induced acute haemolytic anaemia in patients with glucose-6-phosphate dehydrogenase deficiency (G6PDd). Reluctance to use primaquine is reinforced by a lack of quality safety data. This study was conducted to assess the tolerability of a primaquine regimen in Cambodian severely deficient G6PD variants to ascertain whether a weekly primaquine could be given without testing for G6PDd.MethodsFrom January 2013 to January 2014, Cambodians with acute vivax malaria were treated with dihydroartemisinin/piperaquine on days (D) 0, 1 and 2 with weekly doses of primaquine 0.75 mg/kg for 8 weeks (starting on D0, last dose on D49), and followed until D56. Participants’ G6PD status was confirmed by G6PD genotype and measured G6PD activity. The primary outcome was treatment completion without primaquine toxicity defined as any one of: (1) severe anaemia (haemoglobin [Hb] <7 g/dL), (2) a >25 % fractional fall in Hb from D0, (3) the need for a blood transfusion, (4) haemoglobinuria, (5) acute kidney injury (an increase in baseline serum creatinine >50 %) or (6) methaemoglobinaemia >20 %.ResultsWe enrolled 75 patients with a median age of 24 years (range 5–63); 63 patients (84 %) were male. Eighteen patients were G6PDd (17/18 had the Viangchan variant) and had D0 G6PD activity ranging from 0.1 to 1.5 U/g Hb (median 0.85 U/g Hb). In the 57 patients with normal G6PD (G6PDn), D0 G6PD activity ranged from 6.9 to 18.5 U/g Hb (median 12 U/g Hb). Median D0 Hb concentrations were similar (P = 0.46) between G6PDd (13 g/dL, range 9.6–16) and G6PDn (13.5 g/dL, range 9–16.3) and reached a nadir on D2 in both groups: 10.8 g/dL (8.2–15.3) versus 12.4 g/dL (8.8–15.2) (P = 0.006), respectively. By D7, five G6PDd patients (27.7 %) had a >25 % fall in Hb, compared to 0 G6PDn patients (P = 0.00049). One of these G6PDd patients required a blood transfusion (D0–D5 Hb, 10.0–7.2 g/dL). No patients developed severe anaemia, haemoglobinuria, a methaemoglobin concentration >4.9 %, or acute kidney injury.ConclusionsVivax-infected G6PDd Cambodian patients demonstrated significant, mostly transient, falls in Hb and one received a blood transfusion. Weekly primaquine in G6PDd patients mandates medical supervision and pre-treatment screening for G6PD status. The feasibility of implementing a package of G6PDd testing and supervised primaquine should be explored.Trial registrationThe trial was registered on 3/1/2013 and the registration number is ACTRN12613000003774.

Combating Global Antibiotic Resistance: Emerging One Health Concerns in Lower- and Middle-Income Countries

Antibiotic misuse in lower- and middle-income countries (LMICs) contributes to the development of antibiotic resistance that can disseminate globally. Strategies specific to LMICs that seek to reduce antibiotic misuse by humans, but simultaneously improve antibiotic access, have been proposed. However, most approaches to date have not considered the growing impact of animal and environmental reservoirs of antibiotic resistance, which threaten to exacerbate the antibiotic resistance crisis in LMICs. In particular, current strategies do not prioritize the impacts of increased antibiotic use for terrestrial food-animal and aquaculture production, inadequate food safety, and widespread environmental pollution. Here, we propose new approaches that address emerging, One Health challenges.

Low frequency of acute hepatitis E virus (HEV) infections but high past HEV exposure in subjects from Cambodia with mild liver enzyme elevations, unexplained fever or immunodeficiency due to HIV-1 infection.

BACKGROUND In Cambodia, previous studies conducted on hepatitis E virus (HEV) infection are scant, sometimes old, and showed inconsistent results. Moreover, there is no data about HEV infection in Cambodian HIV-1-infected patients. OBJECTIVES To assess the occurrence of acute HEV infections and the level of past HEV exposure in one Mekong country. STUDY DESIGN Using anti-HEV IgM and HEV RNA detection, we retrospectively investigated the presence of acute HEV infection in 825 individuals, including 350 subjects with or without fever, 300 subjects with or without liver enzyme elevations (LEE) and 175 antiretroviral treatment (ART)-naïve, severely immunocompromised HIV-1-infected patients. The detection of anti-HEV IgG was also performed to assess ancient HEV exposure. RESULTS Nine individuals tested positive for anti-HEV IgM yielding an overall rate of 1.1% (95% confidence interval (CI), 0.5-2.0). We did not find significant differences for anti-HEV IgM rates between subjects with unexplained fevers (1.5%) and those with malaria or dengue-associated fever (1.7%) or non-febrile individuals (0%) (P=0.49), and between subjects with (1.5%) and without (2.0%) LEE (P=0.87). No HIV-infected patient tested positive for anti-HEV IgM. HEV RNA was not detected in all tested plasma specimens (n=578). Overall, the anti-HEV IgG prevalence rate was 30.1% (95% CI, 27.0-33.2). CONCLUSIONS The scarcity of recent HEV infection contrasted with the high level of past HEV exposure. The role of HEV in liver disease is likely minor in Cambodia since no HEV RNA was detected in our studied populations, including HIV-positive patients with severe immunodepression.

Aetiology of acute meningoencephalitis in Cambodian children, 2010-2013

Acute meningoencephalitis (AME) is associated with considerable morbidity and mortality in children in developing countries. Clinical specimens were collected from children presenting with AME at two Cambodian paediatric hospitals to determine the major aetiologies associated with AME in the country. Cerebrospinal fluid (CSF) and blood samples were screened by molecular and cell culture methods for a range of pathogens previously associated with AME in the region. CSF and serum (acute and convalescent) were screened for antibodies to arboviruses such as Japanese encephalitis virus (JEV), dengue virus (DENV), and chikungunya virus (CHIKV). From July 2010 through December 2013, 1160 children (one month to 15 years of age) presenting with AME to two major paediatric hospitals were enroled into the study. Pathogens associated with AME were identified using molecular diagnostics, cell culture and serology. According to a diagnostic algorithm, a confirmed or highly probable aetiologic agent was detected in 35.0% (n=406) of AME cases, with a further 9.2% (total: 44.2%, n=513) aetiologies defined as suspected. JEV (24.4%, n=283) was the most commonly identified pathogen followed by Orientia tsutsugamushi (4.7%, n=55), DENV (4.6%, n=53), enteroviruses (3.5%, n=41), CHIKV (2.0%, n=23) and Streptococcus pneumoniae (1.6%, n=19). The majority of aetiologies identified for paediatric AME in Cambodia were vaccine preventable and/or treatable with appropriate antimicrobials. Emerging Microbes & Infections (2017) 6, e35; doi:10.1038/emi.2017.15; published online 24 May 2017

Premier cas de gnathostomose intra-oculaire à Gnathostoma spinigerum, diagnostiqué au Cambodge

RésuméA live intraocular nematode was identified from a 37 year-old man presented with iritis, pain, redness, lacrimation, swelling, vision loss and intermittent blindness during many hours per day of the left eye. By using slit lamp examination, a worm was removed from iris in an ophthalmology outpatient department setting and sent to the Medical Microbiology Laboratory, Institut Pasteur du Cambodge. Gnathostoma spinigerum was identified, based on its typical morphology via microscopic examination. Based on our diagnosis, the patient was treated by oral albendazole and responded well to this therapy.AbstractLa gnathostomose est une helminthose tropicale animale due à un nématode du genre Gnathostoma. Ce genre comprend plusieurs espèces, dont la plus répandue est Gnathostoma spinigerum. Le cycle parasitaire est complexe et comporte au moins 3 hôtes avec la possibilité d’hôtes paraténiques. Le premier hôte intermédiaire est un crustacé (cyclops), le deuxième un poisson d’eau douce ou un batracien, tous les vertébrés peuvent être hôte paraténique, en particulier le porc et les oiseaux. L’Homme se contamine en consommant la chair crue ou peu cuite des hôtes intermédiaires ou paraténiques. Le plus souvent il s’agit de poissons d’eau douce, de porcs, mais aussi d’autres hôtes intermédiaires (serpents, anguilles…) contenant la larve infestante au troisième stade (L3). La contamination par pénétration transcutanée des larves L3 est possible, le temps moyen de pénétration étant de 30 minutes avec un minimum de 6 minutes. Chez l’Homme comme chez les hôtes paraténiques le cycle est bloqué, c’est une impasse parasitaire: la larve L3 « erre » dans les tissus, responsable d’un syndrome de larva migrans cutanée et/ou viscérale. Nous rapportons le premier cas documenté de gnathostomose intra-oculaire survenu au Cambodge. Il s’agit d’un homme de 37 ans présentant des épisodes de cécité de l’oeil gauche, avec douleurs, iritis, larmoiement et oedème. L’examen à la lampe à fente a permis de mettre en évidence une larve de nématode, dont l’aspect était très évocateur de Gnathostoma spinigerum: tête typique en forme d’ampoule avec cinq rangées de petits crochets et fines épines cuticulaires sur sa surface corporelle. La mise en évidence de la larve a permis de faire le diagnostic de certitude. Lorsque la larve n’est pas identifiée, le diagnostic repose le plus souvent sur un faisceau d’arguments épidémiologiques, cliniques et biologiques (hyperéosinophilie, sérodiagnostic positif). Il s’agit du premier cas intraoculaire signalé au Cambodge. Cette pathologie ne doit pas être ignorée car la gnathostomose est une des causes de méningo-encéphalite à éosinophiles. Avant l’utilisation des antiparasitaires, l’atteinte neurologique était compliquée d’une létalité atteignant 25 % et de séquelles à long terme dans 30 % des cas.

Necrotic Tuberculin Skin (Mantoux) Test Reaction: A Case Report and an Estimation of Frequency

Tuberculin skin testing was performed on a 5-year-old girl in Phnom Penh, Cambodia. She had been immunized by Bacille de Calmette et Guérin. She was tested because of a palpable cervical node and a slightly elevated temperature. Within 48 h, a deep necrotic lesion appeared on the volar aspect of the left arm. The lesion was treated locally, and the child was not treated for suspected TB. To our knowledge, this is the first instance of necrosis in 11,392 people who received Tubersol doses since 1996 to date at our International Vaccination Center, for an estimated incidence of 0.18 per 1,000 (95% Poisson 0.04-0.70 per 1,000 doses used). At a follow-up consultation after 77 days, the lesion had scarred and the child showed no signs suggestive of active TB. Although latent TB infection remains the most likely diagnosis, other types of mycobacterial infection may be considered in the tropical setting and in the absence of signs suggestive of active TB.

First reported case of intraocular Gnathostoma spinigerum in Cambodia

RésuméA live intraocular nematode was identified from a 37 year-old man presented with iritis, pain, redness, lacrimation, swelling, vision loss and intermittent blindness during many hours per day of the left eye. By using slit lamp examination, a worm was removed from iris in an ophthalmology outpatient department setting and sent to the Medical Microbiology Laboratory, Institut Pasteur du Cambodge. Gnathostoma spinigerum was identified, based on its typical morphology via microscopic examination. Based on our diagnosis, the patient was treated by oral albendazole and responded well to this therapy.AbstractLa gnathostomose est une helminthose tropicale animale due à un nématode du genre Gnathostoma. Ce genre comprend plusieurs espèces, dont la plus répandue est Gnathostoma spinigerum. Le cycle parasitaire est complexe et comporte au moins 3 hôtes avec la possibilité d’hôtes paraténiques. Le premier hôte intermédiaire est un crustacé (cyclops), le deuxième un poisson d’eau douce ou un batracien, tous les vertébrés peuvent être hôte paraténique, en particulier le porc et les oiseaux. L’Homme se contamine en consommant la chair crue ou peu cuite des hôtes intermédiaires ou paraténiques. Le plus souvent il s’agit de poissons d’eau douce, de porcs, mais aussi d’autres hôtes intermédiaires (serpents, anguilles…) contenant la larve infestante au troisième stade (L3). La contamination par pénétration transcutanée des larves L3 est possible, le temps moyen de pénétration étant de 30 minutes avec un minimum de 6 minutes. Chez l’Homme comme chez les hôtes paraténiques le cycle est bloqué, c’est une impasse parasitaire: la larve L3 « erre » dans les tissus, responsable d’un syndrome de larva migrans cutanée et/ou viscérale. Nous rapportons le premier cas documenté de gnathostomose intra-oculaire survenu au Cambodge. Il s’agit d’un homme de 37 ans présentant des épisodes de cécité de l’oeil gauche, avec douleurs, iritis, larmoiement et oedème. L’examen à la lampe à fente a permis de mettre en évidence une larve de nématode, dont l’aspect était très évocateur de Gnathostoma spinigerum: tête typique en forme d’ampoule avec cinq rangées de petits crochets et fines épines cuticulaires sur sa surface corporelle. La mise en évidence de la larve a permis de faire le diagnostic de certitude. Lorsque la larve n’est pas identifiée, le diagnostic repose le plus souvent sur un faisceau d’arguments épidémiologiques, cliniques et biologiques (hyperéosinophilie, sérodiagnostic positif). Il s’agit du premier cas intraoculaire signalé au Cambodge. Cette pathologie ne doit pas être ignorée car la gnathostomose est une des causes de méningo-encéphalite à éosinophiles. Avant l’utilisation des antiparasitaires, l’atteinte neurologique était compliquée d’une létalité atteignant 25 % et de séquelles à long terme dans 30 % des cas.

Analytical and field evaluation of the Biocentric Generic HCV assay on open polyvalent PCR platforms in France and Cambodia

BACKGROUND Implementation of affordable methods for HCV viremia is a key priority for identifying individuals who need treatment among persons screened positive for HCV antibodies. Different HCV PCR assays for use on open polyvalent PCR platforms are currently commercially available but studies evaluating the performances of these nucleic acid tests are needed. OBJECTIVES In the present study, we evaluated the analytical and clinical performances of a recently developed HCV RNA PCR assay for detection and quantification of HCV viremia. STUDY DESIGN In this study the Biocentric Generic HCV PCR was compared to the Roche Cobas AmpliPrep/Cobas TaqMan HCV RNA assay. Analytical and clinical performances was evaluated on reference materials and HCV plasma samples collected in 141 patients attending at the Montpellier University Hospital in France. Field evaluation was performed on samples collected in 185 patients attending at Medical Laboratory, Institut Pasteur in Cambodia. RESULTS The lower limit of detection ranged from 50 HCV RNA IU/ml to 300 HCV RNA IU/ml using four different Diasorin and Qiagen automated or manual extraction methods. The specificity (CI) and sensitivity of the assay were 100% (92.5-100), and 98.7% (92.3-99.9), respectively, in France, and 100% (95.5-100), and 100% (94.4-100%), respectively, in Cambodia. Bland-Altman analysis shown good agreement between the two assays including for genotypes 6 HCV, which represent the majority of HCV isolates in Cambodia. CONCLUSIONS The Biocentric Generic HCV assay has shown overall satisfactory analytical performances and a close agreement to the Cobas HCV assay on clinical specimens collected in France and Cambodia. There is an urgent need to further evaluate commercial assays dedicated to HCV detection and quantification using open polyvalent PCR platforms in different settings.

Beta-Lactam resistance among Enterobacteriaceae in Cambodia: The four-year itch

Although antibiotics are too often used inappropriately in Cambodia, published data on antimicrobial resistance in this country are scarce. Epidemic dissemination and the transfer of resistance genes to other bacterial species put the population at risk. The aim of this study was to evaluate the frequency and characteristics of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-E) isolated in consecutive samples tested at Institut Pasteur du Cambodge over a 4-year period (2012-2015). Antimicrobial susceptibility testing was performed by disk diffusion on agar technique and the results were read automatically using an OSIRIS system. The Etest was used to determine minimum inhibitory concentrations (MIC) for some resistance phenotypes. The strain most commonly identified was Escherichia coli (63.9%). The proportion of ESBL-E increased gradually over the study period, from 23.8% to 38.4%. ESBL was detected in 42.7% of the E. coli strains and 33.7% of all Klebsiella pneumoniae isolated. The proportion of ESBL-producing E. coli increased significantly from 28.9% in 2012 to 48.2% in 2015, while the increase for K. pneumoniae remained non-significant. Multidrug resistance was high in this Cambodian series, with some strains displaying resistance to all antibiotics available in the country. There is currently no established system for the surveillance of antimicrobial resistance in Cambodia. Collecting samples from clinical settings throughout the country is critical to assess the impact of antimicrobial drug use in patients in Cambodia and in the Mekong Region.