A Case of Plasmodium Malariae Recurrence: Recrudescence Or Reinfection? Romualdo Grande1, Spinello Antinori2,3* , Luca Meroni3, Michela Menegon4 and Carlo Severini4
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Grande et al. Malar J (2019) 18:169 https://doi.org/10.1186/s12936-019-2806-y Malaria Journal CASE REPORT Open Access A case of Plasmodium malariae recurrence: recrudescence or reinfection? Romualdo Grande1, Spinello Antinori2,3* , Luca Meroni3, Michela Menegon4 and Carlo Severini4 Abstract Background: Plasmodium malariae is the most neglected of the six human malaria species and it is still unknown which is the mechanism underlying the long latency of this Plasmodium. Case presentation: A case of PCR-confrmed P. malariae recurrence in a 52-year old Italian man was observed 5 months after a primary attack. In the interval between the two observed episodes of malaria the patient denied any further stay in endemic areas except for a visit to Libya, a country considered malaria-free. Genomic DNA of the P. malariae strain using fve microsatellites (PM2, PM9, PM11, PM25, PM34) and the antigen marker of circumsporozoite (csp) was amplifed and sequenced. Analysis of polymorphisms of the P. malariae csp central repeat region showed dif- ferences between the strains responsible of the frst and second episode of malaria. A diference in the allele size was also observed for the sequence analysis of PM2 microsatellites. Conclusions: Plasmodium malariae is a challenging human malaria parasite and even with the use of molecular techniques the pathogenesis of recurrent episodes cannot be precisely explained. Keywords: Plasmodium malariae, Malaria, Recrudescence, Long-latency Background P. malariae is not a relapsing Plasmodium species, thus Plasmodium malariae is the parasite responsible of quar- still giving the Bignami’s interpretation of endo-eryth- tan malaria with the typical periodicity of fever paroxysm rocytic persistence of the parasite as the more satisfac- observed every 72-h as detailed in a study by Camillo tory [3, 8–10]. However, the fact that the existence of P. Golgi in 1886, but also described in the fourteenth Cen- malariae hypnozoites has never been proven is not “per tury by Dante Alighieri in the Divine Comedy (seventeen se” a proof against it. For instance Plasmodium ovale is Canto of the Inferno) [1–3]. Te parasite is widely distrib- credited to produce hypnozoite although P. ovale hypno- uted in most tropical and sub-tropical areas, with over- zoites have never been demonstrated biologically. Herein lapping presence with Plasmodium falciparum, especially it is described a case of P. malariae infection in an Italian in sub-Saharan Africa, where it might easily be over- man occurring 5 months after a previous malaria episode looked if molecular techniques such as polymerase chain despite the fact he had not travelled to a malaria-endemic reaction (PCR) are not used for diagnosis [1, 4]. Although region. A review of similar cases is also described it is well known that malaria episodes due to P. malariae together with possible explanation of this phenomenon. can occur even after 30–50 years following a previous malaria attack, the mechanism responsible for its persis- Case presentation tence and late recurrence still remains a medical mystery A 52-year-old Italian man sought care at the Emergency [5–8]. Te failure to identify hypnozoites in liver biopsy Department (ED) of Luigi Sacco Hospital in Milan, Italy of either human and animals is considered a proof that on 14 December, 2017, complaining of a quartan pat- tern of fever that started 1 week before together with arthralgia and myalgia. He reported frequent trips to *Correspondence: [email protected] 2 Department of Biomedical and Clinical Sciences “Luigi Sacco”, University sub-Saharan Africa, the last one to Mozambique and of Milan, Milan, Italy several previous malaria attacks treated by himself using Full list of author information is available at the end of the article quinine. He reported to have not taken anti-malarial © The Author(s) 2019. 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. Grande et al. Malar J (2019) 18:169 Page 2 of 9 chemoprophylaxis. A chest X-ray was negative and Table 1 Primers and cycling parameters for PCR laboratory examinations were unremarkable except for Primers Aminoacids PCR cycling an increase of C reactive protein (50.9 mg/L) and mild parameters thrombocytopaenia (153,000/µL). A blood smear was Pm09for ACG ATA ATA ATA TAA ATG GGG 94 °C-30 s, negative for malaria parasites as well as a rapid diagnostic Pm09rev GTT CAT AAC TTT GAT CTT AAC 45 °C-30 s; test (RDT), but species-specifc PCR turned positive for 72 °C-1 min, 40 Pm11for GGG ATA TGA ATT ACA TAC AC cycles P. malariae. He was treated with a standard regimen of Pm11rev CTT TAT TTG TGG TCG AGG oral chloroquine phosphate (1 g per os initially, 500 mg Pm25for CCA AAT AAG TGA CAT ACA AC 6 h after the frst dose, and then 500 mg once a day on Pm25rev GAG GTA ACT TAA AAA ATT CAC the 2nd and 3rd days of therapy). Subsequently he was in Pm02for GGG GCA TAA AGG AAA AAC 94 °C-30 s, good health until the end of April when fever recurred Pm02rev GAA TTT TTG AAT AAC AAG AAA CC 52 °C-30 s; spiking to 40 °C associated with severe headache. On 72 °C-1 min, 40 Pm34for GAA TGG AAA AAT TCC TTC AG 4 May, 2018 he presented to the ED of another hospi- cycles Pm34rev TTG GAC AAT GAA AAA ACT AAG tal where a blood smear was positive for trophozoites Pm MSP1for TTC CAA AAA TTG AGG AAA TGT T of Plasmodium spp. He was transferred to the ED of L. Pm MSP1rev TTT GGA CAA TGT CGG AAC AA Sacco hospital where a new blood smear showed scanty Pm CSfor CCC ACA AAA GCT GTT GAA AA trophozoites of P. malariae; RDT was negative and spe- Pm CSrev TGG TGA CCA TTC CTC CGT A cies-specifc PCR confrmed the diagnosis of P. malariae. Clinical examination was remarkable for the presence of herpes labialis, but otherwise negative. A chest X-ray was negative and blood examinations showed increase sequencing. Te obtained sequences were compiled and C-reactive protein (201 mg/L) mild anaemia (Hb 12.2 g/ analysed by Accelrys DS Gene Software. dL, Ht 35%), leukopaenia (WBC 3200/μL) and thrombo- Comparison of the genetic diversity of P. malariae iso- cytopaenia (45,000/μL). In the period between the two P. lates collected from the patient’s two blood specimens malariae episodes he admitted only a short stay in north- collected on frst hospital admission (14 December, 2017) ern Africa (Libya) without any other trip to sub-Saharan and on second admission (5 May, 2018) was performed Africa. He was admitted to the Infectious Diseases Ward by direct sequencing of the amplifed fragments of six P. and treated with a 3-day course of dihydroartemisinin– malariae molecular markers. piperaquine (320/40 mg) 4 tablet/day for 3 days. He was A PubMed, Scopus and EMBASE literature search discharged on 8 May, 2018 with negative blood smear was performed from 1940 to 2018 with the search terms and PCR for malaria. On follow-up he had normalization P. malariae AND “recrudescence” AND “recurrence” of blood examinations and up to January 2019 no more AND “relapse”. Several cases were added by cross-ref- recurrences of malaria. erencing the articles cited in the retrieved case reports. Articles in Chinese, Russian and Japanese languages Methods were excluded. Plasmodium malariae genomic DNA was extracted from 200 μL of whole infected blood samples collected from Results the patient at both hospital admittances, using PureLink In each of two tested DNA samples, a single amplifed Genomic DNA Kit-Invitrogen. Five microsatellites (MSs) product was observed on agarose gel for each analysed (PM2, PM9, PM11, PM25, PM34) and the antigenic target, suggesting the presence of a single detectable iso- marker P. malariae circumsporozoite (Pmcsp) gene were late for each malaria episode. Te central region of Pmcsp genotyped, by PCR amplifcation and sequencing, in P. gene and three MSs (PM2, PM9, PM34) were successfully malariae isolate(s) responsible for the patient’s infection sequenced in patient’s two blood specimens. Te result of in order to compare the primary infection and the second the sequencing of PM11 and PM25 showed the amplif- episode. cation of non-specifc bands, resulting in a cross-reaction Microsatellite amplifcation was performed using spe- with human DNA, and for this reason these two molecu- cifc primers previously described by Bruce et al. [11], lar markers were excluded by the present analysis. adopting slight modifcations in the amplifcation pro- Analysis of polymorphisms of the Pmcsp central repeat tocol (Table 1). For amplifcation of Pmcsp gene, two region resulted in the amplifcation of a DNA fragment internal primers were designed specifcally and used to of 864 base pairs (bps) (288 aminoacids) in the isolate amplify the central repeat region of the gene (Table 1). responsible of the frst episode, with a repeat region char- All PCR products were examined by gel electrophoresis acterized by two NDAG tetrapeptide repeat units fol- and sent to Eurofns Genomics Company (Germany) for lowed by 51 NAAG tetrapeptide repeat units. Te isolate Grande et al.