MEM INST OSWALDO CRUZ, RIO DE JANEIRO, 103(1) February 2008
PAGES: 39-44 DOI: S0074-02762008000100006 Full paper
Patterns of co-association of C-reactive protein and nitric oxide in malaria in endemic areas of Iran

Hossein NahrevanianI, +; Jafar GholizadehII; Mahin FarahmandI; Mehdi AssmarI

IDepartment of Parasitology, Pasteur Institute of Iran, Pasteur Avenue, 13164 Tehran, Iran
IIUrumia Laboratory of Education and Quality Control, Health Center of West Azerbaijan Province, Urumia, Iran

Abstract

In addition to numerous immune factors, C-reactive protein (CRP) and nitric oxide (NO) are believed to be molecules of malaria immunopathology. The objective of this study was to detect CRP and NO inductions by agglutination latex test and Griess microassay respectively in both control and malaria groups from endemic areas of Iran, including Southeastern (SE) (Sistan & Balouchestan, Hormozgan, Kerman) and Northwestern (NW) provinces (Ardabil). The results indicated that CRP and NO are produced in all malaria endemic areas of Iran. In addition, more CRP and NO positive cases were observed amongst malaria patients in comparison with those in control group. A variable co-association of CRP/NO production were detected between control and malaria groups, which depended upon the malaria endemic areas and the type of plasmodia infection. The percentage of CRP/NO positive cases was observed to be lower in NW compare to SE region, which may be due to the different type of plasmodium in the NW (Plasmodium vivax) with SE area (P. vivax, Plasmodium falciparum, mixed infection). The fluctuations in CRP/NO induction may be consistent with genetic background of patients. Although, CRP/NO may play important role in malaria, their actual function and interaction in clinical forms of disease remains unclear.

Malaria is a world wide problem and it still remains as a concern in the Eastern Mediterranean region and in Iran (Sadrizadeh 1999, WHO 2003). Iran was divided into two malaria zones, North and South of the Zagros Range of Mountains (Manouchehri et al. 1992), including Southeastern (SE) and Northwestern (NW) provinces (Zakeri et al. 2004, Nahrevanian et al. 2006). The prevalent species is Plasmodium vivax followed by Plasmodium falciparum (Zaim 1987, Edrissian 2002) and mixed-infections (Assmar et al. 2003). Out of 23,562 malaria cases have been reported in the country in 2003, more than 70% occurred in the SE part (Zakeri et al. 2002, WHO 2003). Recently, a new threat of imported malaria emerged from the NW part of the country, Parsabad area, which was affected by a serious epidemic of P. vivax (Sadrizadeh 1999). To date, the malaria situation in SE corner is serious (Zakeri et al. 2002). However, in other endemic areas, malaria is hypoendemic with uncomplicated cases (Edrissian et al. 1993). A malaria eradication programme began in Iran in 1949 and changed to malaria control in 1985 as a result of constraints and challenges. In the SE provinces, the major peak of malaria transmission occurs between September and November, with 21% of malaria cases in this region caused by P. falciparum (WHO 2004). Chloroquine (CQ) is still being recommended as the first-line treatment for uncomplicated cases of P. falciparum malaria, with a combination of sulfadoxine-pyrimethamine and quinine recommended for the CQ treatment failures. A combination of CQ and primaquine is the recommended first-line treatment for P. vivaxmalaria (Raeisi et al. 2006). Vector control activities include residual house spraying, larviciding, biological control and insecticide-treated bed nets distribution (WHO 2004).

Increased drug and insecticide resistance has made vaccine preparation urgent for malaria with a focus on new immune targets. Potential mechanisms of immunity against malaria include antibodies, cytotoxic T-cells, cytokines and a variety of other soluble mediators. Macrophages are key components of the antimicrobial immune responses, generating large amounts of toxic molecules, reactive oxygen and nitrogen intermediates (RNI), H2O2and nitric oxide (NO) (Bogdan et al. 2000). In addition to NO, C-reactive protein (CRP) is a major acute phase protein present in normal serum, which increases significantly after most forms of tissue injuries and infections as a non-specific innate defense mechanism of the host. CRP as a protein is mainly regulated at the transcriptional level induced by cytokines (Kremsner et al. 1996, Ablij & Meinders 2002). It is a marker of inflammatory reactions and cytokine activation (Jakobsen et al. 1998), which is produced very early after infection (McCarty et al. 2004). Besides CRP being a marker of the inflammatory process, it may also play a modulating role at the site of inflammation by their effect on the expression of different adhesion molecules. The CRP levels have been increased several times after the acute event with a strong positive correlation between the duration and the intensity of the stimulus and the number of hepatocytes synthesizing CRP (Ablij & Meinders 2002). CRP is reported to be a critical element during malaria infection and there is a strong association of elevated CRP levels during the acute phase of severe (Kremsner et al. 1996) and clinical forms of malaria (Jakobsen et al. 1998).

Although, the role of NO, RNI and NO synthase (NOS) were investigated previously in rodent malaria (Nahrevanian & Dascombe 2001, 2002, Dascombe & Nahrevanian 2003) and human malaria (Nahrevanian et al. 2006), the co-association between NO and other immune factors in clinical forms of malaria remains controversial (Balmer et al. 2000, Cramer et al. 2004). The importance of a balance in the chemokine network was outlined for a protective immunity against malaria infection. The balance includes the amounts of cytokine released, the rate, time and site of production. Interaction, intervention and co-association throughout this network are both interesting and complicated. In addition to NO induction which was reported previously in Iranian malaria patients (Nahrevanian et al. 2006), in this novel immunoepidemiological study, the pattern of co-association between CRP and NO is investigated in malaria patients and control groups from NW and SE endemic malaria regions of Iran.

 

PATIENTS, MATERIALS AND METHODS

Patients and groups - This study has been carried out from 2002 to 2005 to investigate CRP and NO patterns among the malaria patients and control groups from Hormozgan, Sistan & Balouchestan, Kerman (SE) and Ardabil (NW) as malaria endemic provinces (Fig. 1). Two hundreds and thirty five blood samples (55-60/province) were randomly collected from malaria patients referred to the health clinics, who resided in above mentioned endemic areas. In addition, 80 blood samples (20/province) were randomly taken from healthy people with no history of malaria from same endemic regions. Sample selection in both groups was applied considering all statistical parameters including province, gender (control group = male 51%, female 49%; malaria group = male 53%, female 47%) and age groups (control; 30.3 ± 1.9 and malaria; 28.4 ± 1.5). Malaria was confirmed by microscopy and polymerase chain reaction assay (PCR), and all clinical symptoms including fever, tremor, pallor, headache, hepato-splenomegaly and neurological changes were recorded (Table). This study protocol was approved by institutional ethical review board (Ethical Committee of the Pasteur Institute of Iran).

 

 

Blood collection and plasma preparation - 10 ml of peripheral venous blood was collected from malaria patients in two sets of tubes (with EDTA for DNA extraction and with Heparin for CRP and NO assays) as described previously (Nahrevanian et al. 2006).

Malaria diagnosis - Malaria was diagnosed by both microscopy (Giemsa stained thick and thin blood smears) and PCR in patients as previously described (Nahrevanian et al. 2006). Up to 100 microscopic fields were examined to establish the diagnosis.

Plasmodia DNA was extracted from blood samples by nested-PCR as described by Snounou et al. (1993). First round of PCR was carried out using rPLU5 and rPLU6 primers. PCR products were reacted with second round primers including FAL1, FAL2 (P. falciparum), VIV1, VIV2 (P. vivax) and MAL1, MAL2 (Plasmodium malariae) (Snounou et al. 1993). DNA was amplified according to the following condition: denaturing 95ºC/1 min, annealing 58ºC/2 min and extension 72ºC/2 min for 35 cycles. PCR products were run in a 1.5% agarose gel, and observed by UV-transluminator.

Agglutination latex test (ALT) for CRP assay - ALT was based on slide agglutination and applied as recommended by kit manufacturer (SGM, Roma, Italy). Positive and negative control were placed into separate circles on the slide test. The CRP latex reagent was gently swirled and added and mixed to the tested blood sample, and spread over the entire surface of the circle by a mechanical rotator. Latex particles with goat IgG anti-human CRP were agglutinated when mixed with samples containing CRP. The presence or absence of visible agglutination was examined macroscopically and the presence of agglutination indicated a CRP concentration equal or greater than 6 mg/l.

Griess microassay (GMA) - A modified Griess reaction was used (Rockett et al. 1994 modified by Nahrevanian & Dascombe 2001). Standard curves for sodium nitrite and nitrate (Sigma, UK) were prepared. Samples were treated with nitrate reductase (NAD[P]H Aspergillus species, Sigma, UK) and NADPH b-nicotinamide adenine dinucleotide phosphate (Sigma Diagnostics, St Louis, USA). Griess reagent [5% phosphoric acid, 1% sulfanilic acid and 0.1% N (1-naphthyl-1)-ethylendiamine dihydrochloride, all from Sigma, UK, dissolved in 100 ml deionised water] was then added and proteins subsequently precipitated by trichloroacetic acid (BDH, England). Tube contents were mixed then centrifuged (Eppendorf centrifuge 5415 C, Germany) and duplicates of supernatants were transferred to a flat-bottomed microplate, and absorbances read at 520 nm using a microplate reader (Bio-TEK, power wave XS, USA) and values were calculated from standard calibration plots (Nahrevanian & Dascombe 2001).

Statistical analysis - Values are presented as the mean ± SEM for groups of n samples. The significance of differences was determined by Analysis of Variances (ANOVA) and Student's t-test using Graph Pad Prism Software (Graph Pad, San Diego, California, USA) and Microsoft Office Excel 2003.

 

RESULTS

The results indicated that CRP and NO are produced in all malaria endemic areas of Iran. In this study, NO level of patients was compared with that of the related controls in each province, whereas higher NO values rather than average control values were considered as NO positive (NO+) (Nahrevanian et al. 2006). In addition, the presence of agglutination indicated a CRP concentration equal or greater than 6 mg/l as CRP positive (CRP+). Both data from GMA and ALT were calculated and established for a cross comparison amongst study groups. Data revealed more CRP/NO positive cases among malaria patients in comparison to healthy controls. In addition, a higher rate of CRP+ (91.9%) and NO+ (86.4%) cases was observed in SE than NW malaria [CRP+, 12.2%; NO+, 63.3%]. It indicated an association between patterns of CRP+/NO+ cases with geographical distribution of malaria in Iran. This may be related to the different type of malaria in the NW (mostly P. vivax with a minority of mixed infections) and SE regions (P. vivax, P. falciparum and mixed infections) (Table).

Despite of similar patterns of CRP/NO involvement among SE provinces, a different result was found in NW province (p < 0.001). In spite of similar patterns of CRP/NO involvement among SE provinces, a different result was presented in NW province (p < 0.001). A consistent association was observed among all CRP/NO positive and CRP/NO negative cases (Fig. 2). In addition, a relationship was observed between CRP+ cases and the type of malaria infection. The highest association of CRP was observed in P. vivax malaria as a prevalent species, then it was presented in mixed infections of both plasmodia; whereas the lowest involvement was indicated in P. falciparum malaria (p < 0.01, p < 0.001) (Fig. 3). A different variation was detected in CRP and NO values between two control and malaria groups. Variable patterns for CRP (mg/l) and NO (µM) production were represented according to each individual of malaria patients in four endemic provinces. The fluctuations in CRP/NO values induction by different population may be consistent with genetic differences of human profiles (Fig. 4). The results demonstrated an association between CRP+ cases and clinical symptoms mostly fever, tremor and headache, however some variations were observed in provinces including tremor (p < 0.05), pallor (p < 0.001) and headache (p < 0.05) (Fig. 5). A reverse association of CRP positive cases with gender was observed between NW malaria (male 14.3%; female 85.7%: p < 0.001) and SE malaria (male 75.4%; female 24.6%: p < 0.001) regions (Fig. 6A). According to age groups, the CRP+ cases in 1-15 years of malaria patients were 14.3% (NW) and 26.5% (SE); in 16-30 years were 42.8% (NW) and 48.0% (SE); in 31-45 years were 14.3% (NW) and 16.9% (SE) and in over 45 years old were 28.6% (NW) and 8.6% (SE). The major CRP induction was associated with 16-30 years age group in all endemic regions (p < 0.001) (Fig. 6B).

 

 

 

 

 

 

 

 

 


 

DISCUSSION

In this novel immuno-epidemiological study, CRP and NO levels were higher in malaria groups, when compared with controls of same endemic area. The detected high CRP level in malaria in this study is in agreement with previous reports, and it is hence proposed as a useful tool for malaria immuno-epidemiology. The resulting data has revealed a correlation of CRP and NO in malaria patients of NW and SE Iran. Variations arise due to the geographical areas and the type of malaria infection. This may clarify the co-involvement of CRP and NO as two major immune elements during malaria infection in endemic regions of Iran; however it is not justified, whether the CRP/NO production is beneficial or detrimental to the patients. Kremsner et al. (1996) reported the high plasma levels of both CRP and NO in patients with severe malaria than uncomplicated cases. Moreover, stimulated hepatocytes produce CRP, which has an anti-plasmodial effect on the hepatic development of parasite, both by preventing penetration of the sporozoite and by blocking parasite replication (Nussler et al. 1991).

Notwithstanding the conflicting publications, the role of CRP (Kremsner et al. 1996, Gyan et al. 2002) and NO (Awasthi et al. 2003, Clark et al. 2003, Cramer et al. 2004) in the immune responses to Plasmodium remains uncertain. It is suggested that NO alone or in accompany with CRP and/or other chemokines is involved in protective and/or pathogenic responses of human malaria. However, there are substantial data in the literature on CRP and NO suggesting a potent antimicrobial role for both of them individually or acting together. Increased CRP/NO synthesis might have a protective rather than pathological role in malaria (Gillman et al. 2004, Sharma et al. 2004). Moreover, some authors suggested no association with degree of disease and induction of CRP/NO in malaria. These findings did not support a pivotal role for systemic generation of CRP/NO in the pathogenesis of severe malaria (Clark et al. 2003). Some researchers believe that NO (Becker et al. 2004) and CRP or their toxic functions may contribute to the pathology of severe form of disease, and that many of malaria symptoms can be depended on excessive CRP induction (Kremsner et al. 1996, Jakobsen et al. 1998) or NO overproduction (Gyan et al. 2002, Clark et al. 2003). CRP was associated with the pathological signs including fever, splenomegaly, anaemia and cerebral malaria (Gyan et al. 2002). In addition, the duration of symptoms and parasitemia correlated positively with CRP during the acute phase of malaria (Kremsner et al. 1996).

Data of this study emphasized that CRP/NO involvement in malaria varied and depended on endemic regions and strain of Plasmodia or many other unknown factors. Variation of genetic structure and polymorphisms of genes encoding CRP/NO induction could describe the ability of host responses to malaria infection, which are reported in different publications of various regions (Ikeda et al. 2002, Clark et al. 2003, Wells et al. 2005). The variations in CRP/NO induction by different population may be consistent with the human genetic background. The protective polymorphisms may undergo selection in populations with a long history of exposure to malaria and other infections (Boutlis et al. 2003). In addition, population studies generally support an association between protection from severe malaria and CRP/NO production. Genetic epidemiology or cytokine biology alone is not enough to solve CRP/NO paradox, but together they stand a good chance (Gyan et al. 2002, Clark et al. 2003, Mackintosh et al. 2004, Wells et al. 2005).

The higher percentage of NO+ /CRP+ cases in SE provinces may indicate a dependency of NO/CRP induction with type of malaria infections (P. falciparum or mixed infection). However further investigation is needed to clarify this concept.

In conclusion, our data highlighted the fact that CRP/NO are produced in malaria patients of all endemic provincial areas of Iran. The results of published reports clarified the variation rate of CRP/NO production, which were relating to type of infection, provincial regions, species of Plasmodium and clinical symptoms. The present study was not designed to answer questions relating to the role of CRP/NO in protection or pathogenesis during malaria; our findings provided a frame work of immuno-epidemiological investigations between basal CRP/NO production in two healthy and malaria groups in Iran. Although, CRP/NO play an important role in malaria, their actual functions and interactions in clinical forms of disease remain unclear. However, involvement of CRP/NO in malarial host is conflicting (Boutlis et al. 2004, Wells et al. 2005), the complex relationship between malaria symptoms, genetic polymorphisms and CRP/NO production in populations requires further studies to address their immuno-modulatory roles in malaria.

 

ACKNOWLEDGEMENTS

To the Director and the staff from the malaria unit of the Centre for Diseases Control (CDC), Iran, for their cooperation. To the authorities and staff from malaria endemic provinces of Iran during this study. To Dr Vahid Khalaj from Department of Biotechnology, Pasteur Institute of Iran, for reviewing the manuscript and providing helpful suggestions.

 

REFERENCES

Ablij HC, Meinders AE 2002. C-reactive protein: history and revival. Eur J Intern Med 13: 412-422.

Assmar M, Terhovanessian A, Jahani MR, Nahrevanian H, Amirkhani A, Piazak N, Esmaeili AR, Farahmand M, Zare M 2003. Molecular epidemiology of malaria disease in endemic areas of Iran. Southeast Asian J Trop Med Public Health 34: 15-19.

Awasthi A, Kumar A, Upadhyay SN, Yamada T, Matsunaga Y 2003. Nitric oxide protects against chloroquine resistant Plasmodium yoelii nigeriensis parasites in vitro. Exp Parasitol 105: 184-191.

Balmer P, Phillips HM, Maestre AE, McMonagle FA, Phillips RS 2000. The effect of nitric oxide on the growth of Plasmodium falciparum, P. chabaudi and P. berghei in vitro. Parasit Immunol 22: 97-106.

Becker K, Tilley L, Vennerstrom JL, Roberts D, Rogerson S, Ginsburg H 2004. Oxidative stress in malaria parasite infected erythrocytes: host-parasite interactions. Int J Parasitol 34: 163-189.

Bogdan C, Rollinghoff M, Diefenbach A 2000. Reactive oxygen and reactive nitrogen intermediates in innate and specific immunity. Curr Opin Immunol 12: 64-76.

Boutlis CS, Tjitra E, Maniboey H, Misukonis MA, Saunders JR, Suprianto S, Weinberg JB, Anstey NM 2003. Nitric oxide production and mononuclear cell nitric oxide synthase activity in malaria-tolerant Papuan adults. Infect Immun 71: 3682-3689.

Boutlis CS, Weinberg JB, Baker J, Bockarie MJ, Mgone CS, Cheng Q, Anstey NM 2004. Nitric oxide production and nitric oxide synthase activity in malaria-exposed Papua New Guinean children and adults show longitudinal stability and no association with parasitaemia. Infect Immun 72: 6932-6938.

Clark IA, Awburn MM, Whitten RO, Harper CG, Liomba NG, Molyneux ME, Taylor TE 2003. Tissue distribution of migration inhibitory factor and inducible nitric oxide synthase in falciparum malaria and sepsis in African children. Malar J 2: 1-17.

Cramer JP, Mockenhaupt FP, Ehrhardt S, Burkhardt J, Otchwemah RN, Dietz E, Gellert S, Bienzle U 2004. iNOS promoter variants and severe malaria in Ghanaian children. Trop Med Int Health 9: 1074-1080.

Dascombe MJ, Nahrevanian H 2003. Pharmacological assessment of the role of nitric oxide in mice infected with lethal and nonlethal species of malaria. Parasite Immunol 25: 149-159.

Edrissian GH 2002. Malaria history and status in Iran. J Sch Pub Health Inst Pub Health Res 1: 50-60.

Edrissian GH, Afshar A, Sayedzadeh A, Mohsseni G, Satvat MT 1993. Assessment of the response in vivo and in vitro of Plasmodium falciparum to sulphadoxine-pyrimethamine in the malarious areas of Iran. J Trop Med Hyg 96: 237-240.

Gillman BM, Batchelder J, Flaherty P, Weidanz WP 2004. Suppression of Plasmodium chabaudi parasitaemia is independent of the action of reactive nitrogen intermediates and/or nitric oxide. Infect Immun 72: 6359-6366.

Gyan B, Kurtzhals JAL, Akanmori BD, Ofori M, Goka BQ, Hviid L, Behr C 2002. Elevated levels of nitric oxide and low levels of haptoglobin are associated with severe anaemia in African children. Acta Trop 83: 133-140.

Ikeda U, Maeda Y, Yamamoto K, Shimada K 2002. C-reactive protein augments inducible nitric oxide synthase expression in cytokine-stimulated cardiac myocytes. Cardiovas Res 56: 86-92.

Jakobsen PH, McCay V, N'Jie R, Olaleye BO, D'Alessandro U, Zhang G-H, Eggelte TA, Kock C, Greenwood BM 1998. Decreased antitoxic activities among children with clinical episodes of malaria. Infect Immun 66: 1654-1659.

Kremsner PG, Winkler S, Wildling E, Prada J, Bienzle U, Graninger W, Nussler AK 1996. High plasma levels of nitrogen oxides are associated with severe disease and correlate with rapid parasitological and clinical cure in Plasmodium falciparum. Trans Roy Soc Trop Med Hyg 90: 44-47.

Mackintosh CL, Beeson JG, Marsh K 2004. Clinical features and pathogenesis of severe malaria. Trends Parasitol 20: 597-603.

Manouchehri AV, Zaim M, Emadi M 1992. A review of malaria in Iran, (1975-90). J Am Mosq Contr Assoc 8: 381-385.

McCarty MF 2004. AMPK activation may suppress hepatic production of C-reactive protein by stimulating nitric oxide synthase. Med Hypoth 63: 328-333.

Nahrevanian H, Dascombe MJ 2001. Nitric oxide and reactive nitrogen intermediates during lethal and nonlethal strains of murine malaria. Parasite Immunol 23: 491-501.

Nahrevanian H, Dascombe MJ 2002. Expression of inducible nitric oxide synthase (iNOS) mRNA in target organs of lethal and non-lethal strains of murine malaria. Parasite Immunol 24: 471-478.

Nahrevanian H, Gholizadeh J, Farahmand M, Assmar M, Sharifi K, Ayatollahi Mousavi SA, Abolhassani M 2006. Nitric oxide induction as a novel immunoepidemiological target in malaria-infected patients from endemic areas of the Islamic Republic of Iran. Scand J Clin Lab Invest 66: 201-210.

Nussler A, Pied S, Pontet M, Miltgen F, Renia L, Gentilini M, Mazier D 1991. Inflammatory status and preerythrocytic stages of malaria: Role of the C-reactive protein. Exp Parasitol 72: 1-7.

Raeisi A, Ringwald P, Safa O, Shahbazi1 A, Ranjbar M, Keshavarz H, Nateghpour M, Faraji L 2006. Monitoring of the therapeutic efficacy of chloroquine for the treatment of uncomplicated, Plasmodium falciparum malaria in Iran. Ann Trop Med Parasitol 100: 11-16.

Rockett KA, Awburn MM, Rockett EJ, Cowden WB, Clark IA 1994. Possible role of nitric oxide in malarial immunosuppression. Parasite Immunol 16: 243-249.

Sadrizadeh B 1999. Malaria in the world, in the eastern mediterranean region and in Iran. Arch Iran Med 2: 31-37.

Sharma A, Eapen A, Subbarao SK 2004. Parasite killing in Plasmodium vivax malaria by nitric oxide: implication of aspartic protease inhibition. J Biochem 136: 329-334.

Snounou G, Viriyakosol S, Jarra W, Thaithong S, Brown KN 1993. Identification of the four human malaria parasite species in field samples by the polymerase chain reaction and detection of a high prevalence of mixed infections. Mol Biochem Parasitol 58: 283-292.

Wells BJ, Mainous AG, Everett CJ 2005. Association between dietry arginine and C-reactive protein. Nutrition 21: 125-130.

WHO-World Health Organization 2003. A global strategy for malaria control, WHO, Geneva, 30 pp.

WHO-World Health Organization 2004. Report on the second malaria cross-border meeting for Afghanistan, Islamic Republic of Iran and Pakistan. WHO-EM/MAL/311/E/01.05/66 - http://www.emro.who.int/mei/mep/iran.htm.

Zaim M 1987. Malaria control in Iran; Present and future. J Am Mosq Cont Ass 3: 392-396.

Zakeri S, Naimi P, Zare M, Zand Haghighi M, Dinparast-Djadid N 2004. Molecular evidence on changing pattern of mixed Plasmodium falciparum and P. vivax infections during year-round transmission of malaria in Chahbahar, Iran. Irn Biomed J 8: 89-93.

Zakeri S, Talebi-Najafabadi S, Zare A, Dinparast-Djadid N 2002. Detection of malaria parasites by nested PCR in south-eastern, Iran: Evidence of highly mixed infections in Chahbahar district. Malar J 1: 1-6.

Received 12 June 2007
Accepted 26 December 2007
Financial support: Pasteur Institute of Iran (project n. 200)

+ Corresponding author: This e-mail address is being protected from spambots. You need JavaScript enabled to view it.

CONTACT US

Memórias do Instituto Oswaldo Cruz

Av. Brasil 4365, Castelo Mourisco
sala 201, Manguinhos, 21040-900
Rio de Janeiro, RJ, Brazil

Tel.: +55-21-2562-1222

This e-mail address is being protected from spambots. You need JavaScript enabled to view it.

twitterfacebook

SUPPORT PROGRAM

marca fiocruzmarca brasil
marca faperjmarca cnpqmarca capes n marca cope

and diabetes. Erection dysfunction or ED is certainly one of mens most usual problem. It changes buy tadalafil 60mg A common drug is actually an imitation of its manufacturer twin. Both ought to be same in female cialis 20mg Long Phrase Viagra Use Fundamentally Damages Sex Lives This discount cialis canada Equally so, theres something to be said for the wonder of the second, captured forever on picture or a buy cheap cialis People extremely annoyed that they could only get three weeks at a time, Bunker noted. Retired persons cheap pharmacy These types of matters are possibly to being identified as having a result of cancer buy cialis 40mg - Yoghourt - fat-free simply Physical causes: Buying generic medicines now has been cheap generic cialis Herbaceous plants like nigrum and tribulus are well-known for his or her qualities in defeating impotence, which tadalafil 10mg It is not hard to consider Cialis that is generic. Most men start with one-10 mg dosage each purchase cialis Tadalafil quickly gained the moniker of weekender in Paris due to the fabulous results. The bash freaks buy female cialis