The significance of molecular diagnostics in the evaluation of the clinical course and outcome of malaria

Uvod: Malarija je najznaajnija parazitska infekcija u svetu. Procena je da je 2012. godine bilo 207 miliona sluajeva malarije sa 627.000 smrtnih ishoda u endemskim podrujima, posebno u tropskim i subtropskim regionima (97 zemalja). Brza i pouzdana dijagnoza malarije predstavlja osnovu za primenu odgovarajue terapije i povoljan terapijski ishod. Zlatni standard u dijagnostici malarije je još uvek mikroskopija, mada je s jedne strane subjektivna a s druge, posebno u odreenim klinikim situacijama, nedovoljno osetljiva metoda. Otuda je veliki pomak u dijagnostici predstavljalo uvoenje molekularnih tehnika, za koje je pokazano da imaju veu osetljivost i specifinost, ali nije do kraja jasan njihov stvarni kliniki znaaj. Cilj: Cilj ovog rada je procena klinikog znaaja real-time PCR-a (qPCR) u dijagnostici malarije, posebno u sluaju submikroskopske malarije, kao i da se ispita klinika upotrebljivost praenja DNK-nemije za procenu efekta antimalarine terapije. Metodologija: U istraživanje je ukljueno 109 ispitanika koji su boravili u malarinim podrujima, kod kojih je dijgnostik mlrije vršena n Odseku z przitologiju na Klinici za infektivne i tropske bolesti u Beogradu od jul 2010. do mj 2013. godine. Kod svih ispitanika prvo je raena mikroskopija a potom skrining qPCR gde je kao ciljni gen korišen visoko konzervirani region 18S rRNK gena. Pozitivni uzorci su potom ispitivani specijes-specifinim qPCR-om za identifikaciju etiri vrste roda Plasmodium: P. falciparum, P. vivax, P. ovale i P. malariae. Rezultati: Malarija je dijagnostikovana kod 45 simptomatskih bolesnika, od kojih je kod 42 mikroskopski potvrena. Kod ostala tri (6,7%) bolesnika, dijagnoza je postavljena indirektno, na osnovu adekvatnog terapijskog odgovora...population in 97 countries. In 2012, 207 million cases and 627.000 deaths occurred in malaria-endemic regions, concentrated in the tropics and subtropical areas. A prompt diagnosis with accurate identification of species is crucial for adequate treatment. Conventional microscopic diagnosis, although still the gold standard, is highly subjective, depending on the skill of the microscopist. This has been overcome by molecular methods, which are constantly being improved for increased sensitivity and specificity. Objective: This study aimed to investigate the use of quantitative (q) real-time PCR as a confirmatory method for the diagnosis of malaria in diagnostically uncertain cases. Further aims included investigation of whether qPCR could be used for quantification of parasite density and for how long parasite DNA is detectable in patients receiving antimalarial therapy. Methodology: The study group included 109 individuals after their return from tropical and subtropical areas, tested for malaria in the Parasitological Laboratory at the Hospital for Infectious and Tropical Diseases in Belgrade between July 2010 and May 2013. Patient blood samples were first examined by microscopy and tested for the presence of the parasite 18S rRNA gene by screening qPCR. All positive samples were subsequently analysed by species-specific qPCR for the detection of four Plasmodium species, including P. falciparum, P. vivax, P. ovale and P. malariae. Results: Malaria was diagnosed in 45 patients, all of which were clinically suspected of malaria. In 42 (93.3%), the diagnosis was based on microscopy, and three (6.7%) were patients with submicroscopic malaria (SMM), in whom the diagnosis was based on the favorable effect of antimalarials administered in clinically suspected patients..

Human case of fasciolosis in Serbia treated with triclabendazole

Introduction. The number of humans infected by Fasciola hepatica is increasing worldwide. Humans can become accidental hosts by ingesting drinking water or plants contaminated with metacercariae. Case report. We reported a case of a 68-year-old Serbian woman, in which the diagnosis of acute fasciolosis had been established after serious diagnostic concerns. Based on clinical picture (episodic right upper quadrant abdominal pain, febrility and generalized body pain) and biochemical analyses (high eosinophilia and high activity of alkaline phosphatase), she was appointed as suspected to the acute fasciolosis. Stool and duodenal aspirate exams were negative for Fasciola ova. In the absence of adequate serologic diagnostic for fasciolosis in Serbia, the diagnosis was confirmed using enzyme immunoassays and immunoblot at the Institute for Tropical Diseases in Hamburg, Germany. Soon after triclabendazole was administered, the symptoms disappeared and biochemical values returned to normal. Conclusion. The diagnosis of human fasciolosis may be problematic and delayed, especially in non endemic areas, because physicians rarely encounter this disease and a long list of other diseases must be considered in the differential diagnosis. The syndrome of eosinophilia, fever, and right upper quadrant abdominal pain suggest acute fasciolosis. Unclear source does not rule out fasciolosis

Increased tumor necrosis factor alpha and interleukin-6 serum levels and their correlation with laboratory parameters in patients with imported malaria

In malaria, blood concentrations of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6, are increased. In a study which included 34 patients, TNF-α and IL-6 were examined in two phases, immediately after the admission of patients, and at the end of antimalarial therapy, when the parasitemia was negative. The results show a significant increase of TNF-α and IL-6 in the first phase, before the effects of antimalarial therapy. A very strong correlation between TNF-α and IL-6 is also confirmed, which suggests their coordinated production. Increased TNF-α values were correlated with an older age, the level of parasitemia, the number of platelets and leukocytes, elevated values of procalcitonin, D-dimer and lactate dehydrogenasе, and lower values of serum iron and antithrombin. Increased values of IL-6 were correlated with the level of parasitemia, the number of platelets and leukocytes, and elevated values of D-dimer and lactates

Ekologija komaraca roda Anopheles na području Beograda u proceni vektorskog potencijala za ponovno uspostavljanje transmisije malarije

Belgrade is situated in the area that is potentially at risk from malaria outbrakes. Until eradication, the main vector of malaria in this area was Anopheles maculipennis s. s. (previous name An. typicus) and secondary vectors were An. messeae and An. atroparvus. In this study we examined the distribution and ecology of Anopheles mosquitoes (Diptera, Culicidae) in Belgrade. Females of Anopheles mosquitoes were collected from animal shelters in Belgrade at eight locations during 2003. Egg morphology was used to identify the specimens. A total of 3704 females deposited eggs ready for identification. Three species of An. maculipennis complex were identified: An. messeae, An. atroparvus and An. maculipennis s. s.. The most abundant species were An. messeae (64%). The relative frequency of three species varied depending on the site of collection. Seasonal fluctuations of mosquitoes' species varied. Each develops in a distinct type of water, too. The three species of the An. maculipennis complex, particularly An. messeae and An. atroparvus, are considered as potential vectors of malaria in Belgrade. With the possible reintroduction of Plasmodium species due to climatic changes and increased travel to and from the countries where malaria is endemic, a more efficient vector control is necessary.Beograd je smešten u području koje je potencijalno rizično za ponovno uspostavljanje transmisije malarije. Do eradikacije malarije, glavni vektor malarije na području Beograda bio je An. maculipennis s. s. (raniji naziv An. typicus) a sekundarni vektori bili su An. messeae i An. atroparvus. Mi smo analizirali distribuciju i ekologiju komaraca roda Anopheles (Diptera, Culicidae) na području Beograda. Ženke komaraca roda Anopheles sakupljali smo tokom 2003. godine na 8 lokaliteta šireg područja Beograda. Identifikaciju vrsta vršili smo na osnovu morfologije položenih jaja. Od ukupnog broja izlovljenih ženki komaraca roda Anopheles, njih 3704 je položilo jaja, a njihovom identifikacijom nađene su tri vrste komaraca roda Anopheles, svi pripadnici Anopheles maculipennis kompleksa: An. messeae, An. atroparvus i An. maculipennis s. s.. U ukupnoj populaciji najzastupljeniji je bio An. messeae 64%, zatim An. atroparvus 21%, a najmanje Anopheles maculipennis s. s. 8%. Postojala je razlika u procentualnoj zastupljenosti ovih vrsta u ukupnoj Anopheles populaciji prema lokalitetima, po mesecima, prema izboru vodenih staništa. Prisutne vrste Anopheles komaraca, posebno An. messeae i An. atroparvus ukazuju da je Beograd receptivan za transmisiju malarije, a klimatski uslovi tokom leta pogodni su za kompletiranje sporogoničnog razvoja pripadnika roda Plasmodium

Seasonality of trichinellosis in patients hospitalized in Belgrade, Serbia

A retrospective study of the course and outcome of trichinellosis in a series of 50 patients hospitalized at the Institute for Infectious and Tropical Diseases in Belgrade between 2001 and 2008 was performed. Clinical diagnosis of trichinellosis was based upon the patients' clinical history, symptoms and signs, and eosinophilia. The occurrence of cases showed a strong seasonality (P lt 0.00011. The incubation period ranged between one and 33 days. The mean time between onset of symptoms and admission was nine days. Family outbreaks were the most frequent. Smoked pork products were the dominant source of infection (76 %). Fever was the most frequent clinical manifestation (90 %), followed by myalgia (80 %) and periorbital edema (76 %). 43 patients were examined serologically and 72 % of them had anti-Trichinella antibodies. Eosinophilia and elevated levels of serum CK and LDH were detected in 94, 50 and 56 % of the patients, respectively. All patients responded favorably to treatment with mebendazole or albendazole, but eight developed transient complications. Trichinellosis remains a major public health issue in Serbia

Taeniosis and cysticercosis in Serbia, 1990-2018: Significance of standard of living

Objectives: As is the case for all of Southeast Europe, Serbia is an area traditionally endemic for Taenia saginata and Taenia solium infections. This study was performed to analyse the epidemiological data on taeniosis and cysticercosis in Serbia for the period 1990-2018. Methods: Data on cases of T. saginata and T. solium infection were collected via a systematic search of published articles, the grey literature, and official reports, as well as by performing clinical observational studies of patients treated in the departments for infectious diseases of hospitals and university clinics in Serbia. Results: A total of 212 cases of taeniosis were reported, all between 1997 and 2004 when taeniosis was notifiable (incidence range 0.04-0.9/100 000 population/year). From 1990 to 2018, 170 cases of cysticercosis (all but one of neurocysticercosis), were registered (incidence range 0-0.29/100 000 population/year), with a strong decrease since 2000 and a single case in the last 9 years. The annual number of cases of both taeniosis (Pearson's r = 0.914, p = 0.001) and cysticercosis (Pearson's r = 0.582, p = 0.014) correlated with the consumer price index. Conclusions: In Serbia, T. saginata and T. solium infections are autochthonous but occur only sporadically. However, the potential for re-emergence exists, depending on the socio-economic state of the country

Epidemiologija i dijagnostika fascioloze ljudi

Fasciolosis is a zoonotic infection caused by the trematoda Fasciola hepatica and Fasciola gigantica. Human fasciolosis is endemic in some parts of South America, Africa, Eastern Asia and Europe. High prevalence of human fasciolosis does not necessarily occur in areas where fasciolosis is a major veterinary problem. Infection with Fasciola hepatica has not been reported in humans in Serbia and former Yugoslavia, although a large variety of animals, such as sheep and cattle show infection rates that may reach significant proportions in some areas. Humans can become accidental hosts of this parasite by ingesting contaminated drinking water or plants in an endemic area. Infection with Fasciola hepatica has a variable clinical presentation depending on the stage of the disease. Typical symptoms that may be associated with fascioliasis can be divided by the phases of the disease including the acute or liver phase, the chronic or biliary phase, the obstructive phase, and ectopic or pharyngeal fascioliasis. The diagnosis of human fasciolosis may be problematical and delayed, especially in non-endemic areas, because physicians rarely encounter this disease and a long list of other diseases must be included in the differential diagnosis. The diagnosis of fasciolosis is complex and requires the application of direct an indirect methods of diagnostics: clinical diagnosis, haematological and biochemical findings, parasitological diagnosis, immuno-diagnosis, imaging procedures, liver biopsy. At the Clinic for Infectious and Tropical Diseases in 2005, we recorded the first case of human fasciolosis in a woman from Belgrade, a citizen of Serbia, who developed clinical symptoms of acute fasciolosis after several months of living in Bosnia-Herzegovina. This article reviews the epidemiology and diagnostics of human fasciolosis. .Fascioloza je zoonozna infekcija izazvana trematodama Fasciola hepatica i Fasciola gigantica. Fascioloza ljudi je endemska bolest u pojedinim delovima Južne Amerike, Afrike, istočne Azije i Evrope. Visoka prevalencija fascioloze ljudi nije uvek povezana sa visokom prevalencijom animalne fascioloze. Iako je fascioloza domaćih životinja, pre svega ovaca i goveda, značajno prisutna u pojedinim krajevima Srbije i bivše Jugoslavije, nema dokumentacije, odnosno objavljenih slučajeva fascioloze ljudi u Srbiji. Čovek postaje slučajan domaćin unošenjem infektivnih oblika parazita kontaminiranom vodom ili biljkama. Klinička slika ove bolesti je raznovrsna i zavisi od stadijuma infekcije: akutna ili hepatična, hronična ili bilijarna, obstruktivna, ređe ektopična ili faringealna. Dijagnostika fascioloze ljudi može da bude komplikovana i dugotrajna, naročito u krajevima gde se bolest retko javlja i gde se na nju retko pomišlja, kao i zbog brojnih bolesti slične kliničke manifestacije, koje treba isključiti. Dijagnostika fascioloze ljudi je kompleksna i podrazumeva primenu direktnih i indirektnih dijagnostičkih procedura. Dijagnoza se zasniva na kliničkoj slici, hematološkim i biohemijskim nalazima, parazitološkom nalazu, imunološkoj dijagnostici, tehnikama vizuelizacije patoloških promena i biopsiji jetre. Na Klinici za infektivne i tropske bolesti 2005. godine je dijagnostikovan prvi slučaj fascioloze ljudi u Srbiji. Pacijentkinja iz Beograda razvila je kliničku sliku akutne fascioloze nakon nekoliko meseci boravka u Hercegovini. Ovaj rad predstavlja osvrt na epidemiološki i dijagnostički aspekt fascioloze ljudi.

Transplantation-related risk of Toxoplasma gondii infection: the National Reference Laboratory prospective cohort study results

Toksoplazmoza je česta ali kod pacijenata lečenih transplantacijom uglavnom zanemarena i pogrešno dijagnostikovana oportunistička infekcija koja može ugroziti engraftment ali može i evoluirati u životno ugrožavajuću diseminovanu infekciju. Nakon transplantacije, infekcija parazitom Toxoplasma gondii se može razviti kao reaktivacija hronične infekcije ili može biti preneta graftom. Naša osmogodišnja prospektivna studija bila je usmerena na dijagnostiku i monitoring toksoplazmatske infekcije (TI) kod primalaca matičnih ćelija hematopoeze (haematopoietic stem cell transplant, HSCT) u centru koji primenjuje protokol uzdržavanja od profilakse do engraftmenta, i kod primalaca transplantata srca (heart transplant, HT) koji su na kontinuiranoj profilaksi trimetoprim- sulfametoksazolom (TMP-SMX). Cilj nam je bio utvrđivanje incidence TI u ova dva vrlo različita transplantaciona režima, i to pre nego što evoluira u klinički manifestnu, potencijalno fatalnu bolest (Toxoplasma disease, TD). Pre-transplantacioni serološki i qPCR skrining u post-transplantacionom toku zamenjen je redovnim qPCR monitoringom iz uzoraka periferne krvi (peripheral blood, PB) usmerenim na Toxoplasma 529 bp gen. Kod primalaca HSCT, qPCR je rađen jednom nedeljno dok je kod primalaca HT qPCR rađen jednom mesečno prva dva meseca post-HT i potom jednom godišnje. TI je dijagnostikovana na bazi pozitivnog PCR rezultata iz bar jednog uzorka PB. TI je dijagnostikovana kod 21/104 (20.2%) primalaca HSCT, prevashodno nakon alogene (19/75) i retko nakon autologne HSCT (2/29). Više od 50% slučajeva TI dijagnostikovano je tokom prvog meseca post-HSCT, pre engraftmenta odnosno tokom uzdržavanja od profilakse. Sa druge strane, TI je dijagnostikovana kod 3/37 (8.1%) primalaca HT. Uprkos primeni TMP-SMX, qPCR je postao pozitivan godinu dana posle HT kod dva i dve godine post-HSCT kod trećeg pacijenta. Infekcija je bila preneta graftom kod 2/3 (seronegativni) a reaktivirana kod 1/3 primalaca HT (seropozitivni primalac HT poreklom od seropozitivnog donora). Naši rezultati potvrđuju da je sistemski qPCR monitoring iz uzoraka PB dragocen u dijagnostici TI ne samo kod primalaca HSCT već i kod primalaca solidnih organa, posebno nakon HT. Učestalost qPCR monitoringa se mora adaptirati shodno specifičnostima transplantacionog protokola, pre svega primeni profilakse ali i osnovnoj dijagnozi, na način koji omogućava pravovremenu primenu specifične terapije u svakom slučaju TI.Toxoplasmosis is a common but often neglected and misdiagnosed opportunistic infection in transplant recipients, which can not only compromise the engraftment, but also evolve into life-threatening disseminated infection. Post-transplantation, Toxoplasma gondii infection can develop as a reactivation of chronic infection or could be graft-transmitted. We conducted an eight-year-long prospective study on the diagnosis and monitoring of Toxoplasma infection (TI) in haematopoietic stem cell transplant (HSCT) recipients in a setting that withholds prophylaxis until engraftment, and in heart transplant (HT) recipients on continuous trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis. The objective was to determine the incidence of TI before it evolves into clinical, potentially fatal Toxoplasma disease (TD), in these two very different transplantation settings. Pre-transplantation serological and qPCR screening was followed by post-transplantation peripheral blood (PB)-based qPCR monitoring targeting the Toxoplasma 529 bp gene. In HSCT recipients, qPCR was performed weekly while in HT recipients, qPCR was performed monthly for two months post-HT and then yearly. TI was diagnosed based on a positive PCR result in at least one PB sample. TI was diagnosed in 21/104 (20.2%) HSCT recipients, predominantly after allogeneic (19/75) and rarely after autologous HSCT (2/29). Over 50% of TI cases were diagnosed during the first month post-HSCT, while awaiting engraftment without prophylaxis. On the other hand, TI was diagnosed in 3/37 (8.1%) HT recipients. Regardless of the TMP-SMX prophylaxis, qPCR became positive one year after HT in two and two years post-HSCT in third patient. Infection was graft-transmitted in 2/3 (seronegative) and reactivated in 1/3 OHT (seropositive recipient of a seropositive donor’s heart transplant). The presented results show that systematic PB-based qPCR monitoring is a valuable resource for the diagnosis of TI not only in HSCT but also in solid organ recipients, especially after HT. Frequency of qPCR monitoring should be adjusted according to the specificity of the transplantation setting, especially in terms of prophylaxis but also an underlying diagnosis, in a manner allowing for prompt introduction of specific treatment in each case of TI

The significance of molecular diagnostics in the evaluation of the clinical course and outcome of malaria

Uvod: Malarija je najznaajnija parazitska infekcija u svetu. Procena je da je 2012. godine bilo 207 miliona sluajeva malarije sa 627.000 smrtnih ishoda u endemskim podrujima, posebno u tropskim i subtropskim regionima (97 zemalja). Brza i pouzdana dijagnoza malarije predstavlja osnovu za primenu odgovarajue terapije i povoljan terapijski ishod. Zlatni standard u dijagnostici malarije je još uvek mikroskopija, mada je s jedne strane subjektivna a s druge, posebno u odreenim klinikim situacijama, nedovoljno osetljiva metoda. Otuda je veliki pomak u dijagnostici predstavljalo uvoenje molekularnih tehnika, za koje je pokazano da imaju veu osetljivost i specifinost, ali nije do kraja jasan njihov stvarni kliniki znaaj. Cilj: Cilj ovog rada je procena klinikog znaaja real-time PCR-a (qPCR) u dijagnostici malarije, posebno u sluaju submikroskopske malarije, kao i da se ispita klinika upotrebljivost praenja DNK-nemije za procenu efekta antimalarine terapije. Metodologija: U istraživanje je ukljueno 109 ispitanika koji su boravili u malarinim podrujima, kod kojih je dijgnostik mlrije vršena n Odseku z przitologiju na Klinici za infektivne i tropske bolesti u Beogradu od jul 2010. do mj 2013. godine. Kod svih ispitanika prvo je raena mikroskopija a potom skrining qPCR gde je kao ciljni gen korišen visoko konzervirani region 18S rRNK gena. Pozitivni uzorci su potom ispitivani specijes-specifinim qPCR-om za identifikaciju etiri vrste roda Plasmodium: P. falciparum, P. vivax, P. ovale i P. malariae. Rezultati: Malarija je dijagnostikovana kod 45 simptomatskih bolesnika, od kojih je kod 42 mikroskopski potvrena. Kod ostala tri (6,7%) bolesnika, dijagnoza je postavljena indirektno, na osnovu adekvatnog terapijskog odgovora...population in 97 countries. In 2012, 207 million cases and 627.000 deaths occurred in malaria-endemic regions, concentrated in the tropics and subtropical areas. A prompt diagnosis with accurate identification of species is crucial for adequate treatment. Conventional microscopic diagnosis, although still the gold standard, is highly subjective, depending on the skill of the microscopist. This has been overcome by molecular methods, which are constantly being improved for increased sensitivity and specificity. Objective: This study aimed to investigate the use of quantitative (q) real-time PCR as a confirmatory method for the diagnosis of malaria in diagnostically uncertain cases. Further aims included investigation of whether qPCR could be used for quantification of parasite density and for how long parasite DNA is detectable in patients receiving antimalarial therapy. Methodology: The study group included 109 individuals after their return from tropical and subtropical areas, tested for malaria in the Parasitological Laboratory at the Hospital for Infectious and Tropical Diseases in Belgrade between July 2010 and May 2013. Patient blood samples were first examined by microscopy and tested for the presence of the parasite 18S rRNA gene by screening qPCR. All positive samples were subsequently analysed by species-specific qPCR for the detection of four Plasmodium species, including P. falciparum, P. vivax, P. ovale and P. malariae. Results: Malaria was diagnosed in 45 patients, all of which were clinically suspected of malaria. In 42 (93.3%), the diagnosis was based on microscopy, and three (6.7%) were patients with submicroscopic malaria (SMM), in whom the diagnosis was based on the favorable effect of antimalarials administered in clinically suspected patients..

Ekološko razaranje poljoprivrednih resursa kao posledica ratnih dejstava

In a desperate attempt to enhance and justify his existence and development man has seriously disturbed the natural equilibrium thus imperiling his future on the planet. Scientific and technological revolution has left its marks on the production of inconceivable accurate equipment and weapons intended directly for man's extinction in war conflicts and indirectly to the pollution-contamination of soil, water, air and major natural and agricultural resources. Recent war conflicts in Bosnia and Herzegovina and Republic Srpska with the objective of changing once again the already firmly established borders between countries, took numerous precious lives and provoked enormous destructions and damages but most of all contributed to long-term ecological destruction of some major resources of agricultural production.Čovek je u trci za svoj opstanak i razvoj, tako ozbiljno poremetio prirodnu ravnotežu, da je počeo da dovodi u pitanje svoju budućnost. Naučno-tehnološka revolucija je pokazala svoje tragove u proizvodnji skoro nezamislivih savršenih ratnih oruđa, opreme i drugo koja je opet stvorena da uništi čoveka direktno u ratnim sukobima i indirektno zagađivanjem kontaminacijom zemljišta, voda, vazduha i drugih značajnih prirodnih, pa i poljoprivrednih resursa. Najnovija ratna dejstva na području Bosne i Hercegovine, Republike Srpske potpomognuta nastojanjima za ponovnu teritorijalnu podelu sveta, pored brojnih ljudskih žrtava i ogromnih materijalnih šteta, odrazila su se i na ekološko narušavanje važnijih resursa za poljoprivrednu proizvodnju