A clinical report on mixed infection of malaria, dengue and chikungunya from New Delhi, India

Abstract

Arthropod-borne infections like malaria, dengue and chikungunya fever are transmitted by mosquitoes. The present report describes an unusual case of mixed infection with malaria, dengue and chikungunya viruses in a 21 year old male patient from New Delhi, India during monsoon season of 2016. The malarial fever was diagnosed by thin slide microscopy and antigen test. Chikungunya virus IgM was detected in the sample by the card test. Dengue and chikungunya viruses were further confirmed by RT-PCR for CprM and E1 gene respectively. Phylogenetic analysis clustered the study dengue virus serotype 3 sequence in the genotype III. Thus, the mono infections can not be differentiated from concurrent infections on the basis of clinical symptoms, the appropriate laboratory diagnosis is essential for the accurate pathogen confirmation. Precise and appropriate identification of the multiple pathogens in such clinical cases will assist in the effective management of these arthropod mediated infections.

Electronic supplementary material

The online version of this article (doi:10.1007/s13337-017-0404-6) contains supplementary material, which is available to authorized users.

Arthropod-borne infections like dengue, chikungunya and malaria, transmitted by mosquitoes are of great public health concern in India and other tropical and subtropical region of the world. Dengue (DENV) and chikungunya viruses (CHIKV) are transmitted to humans by the common vector i.e. Aedes mosquito [5, 6]. Malaria fever is caused by the parasite (Plasmodium species) and is transmitted to humans by the infected mosquito Anopheles. All these infections are distributed in common geographical regions and show similar signs and symptoms that become a barrier for the accurate identification of these pathogens. Concurrent infections with dengue and chikungunya viruses have been reported from different geographical regions including India [1, 4, 6, 8]. Mixed infections of Plasmodium with DENV or CHIKV have been reported in different studies [2, 3, 10, 11]. But, concurrent infection with parasite and viruses is rarely documented. An investigation reported concurrent infection with Plasmodium, DENV and CHIKV in a traveler from India [14]. Other reports also showed mixed infection with three pathogens from India [15]. The present study describes such case of mixed infection with three pathogens transmitted by mosquitoes from New Delhi, India during monsoon season 2016.

This sample was collected during an ongoing study of epidemiological analysis of dengue and chikungunya viruses in our laboratory. The study protocol was approved by the Institutional Ethics Committee, Jamia Millia Islamia. Written informed consent was obtained from the patient before sample collection. On 6th September 2016, an adult male student, 21 years of age from Jamia Millia Islamia, New Delhi residing in the nearby locality visited the Out Patient Department (OPD) of University Health Centre. He presented with the symptoms of high grade fever with shivering since last 6–8 days. In addition, he also complained of constipation, headache, weakness and loss of appetite. Rashes and hemorrhagic tendencies were not observed. His two roommates were also suffering from high grade fever since last few days. Blood sample (acute phase) was collected and processed for routine laboratory investigations including haemogram, malarial antigen (Malaria Antigen Kit SD BIOLINE, Alere, Korea), peripheral smear for malaria parasite and Widal test for typhoid fever. Additionally, RT-PCR and IgM lateral flow immunoassay for dengue virus (OnSite Dengue IgG/IgM 3.0 Combo Rapid Test, CTK Biotech) and chikungunya virus [OnSite Chikungunya IgM Combo (CTK) Rapid test, CTK Biotech] was also performed keeping in view of co-circulation of these viruses during that time in this region.

The findings of the laboratory investigations suggested that red cell indices of the patient were almost within normal limits with slightly low hemoglobin. Malaria was found positive by both the methods of thin slide microscopy and by Rapid Malaria Antigen Kit. Widal titer was < 1:40 for typhoid. So, the patient was diagnosed with malaria fever on the basis of clinical symptoms and initial laboratory investigations. The laboratory reports were handed over to the patient on the same day. Therefore, the primary treatment was prescribed to the patient on the same day with recommended anti malaria dose, paracetamol and antacid gel. After his first visit to the Health Centre, the patient went to his permanent residence in Mewat, Haryana, India so further follow up was not recorded.

Large number of cases of dengue and chikungunya virus were reported during monsoon season of 2016 from Delhi [13]. The sample was therefore later (within next 2–3 days) tested for DENV and CHIKV IgM by rapid card test and for viral RNA by RT-PCR at the Virology Laboratory. The sample was negative for DENV IgM, but was positive for CHIKV IgM. Further, the sample was found to be positive for both the dengue and chikungunya viruses by RT-PCR using previously reported primers [1]. All the reactions were carried out with positive and negative controls to avoid contamination. An amplicon of 292 bp specific for CprM region of DENV-3 was obtained from the sample by RT-PCR. The DENV sequence was also confirmed by commercial DNA sequencing followed by BLAST (GenBank Accession number KY099619). Phylogenetic analysis suggested that the study DENV-3 sequence (292 bp) clustered in the genotype III (Fig. 1). The study DENV strain showed close similarity with the sequences from India, and Pakistan. Further, 852 bp amplicon specific for CHIKV E1 envelope gene was also obtained from the sample. However, the CHIKV strain could not be sequenced due to inadequate clinical sample. It may be noted that the PCR was repeated twice for each pathogen from the RNA extraction step to confirm the two viruses.

Fig. 1.

Maximum likelihood tree based on the partial sequence of CprM of dengue virus 3. The tree was constructed using Tamura Nei model with 1000 bootstrap value using MEGA 6.0. The analysis involved 25 nucleotide sequences. The study DENV-3 sequence (red color) clustered with Genotype III (color figure online)

Mosquito-borne infections such as malaria, dengue and chikungunya fever are reported from different geographical regions. But concurrent infections with these pathogens are rarely reported in literature probably due to overlapping symptoms like fever, rashes, headache, vomiting, nausea, muscle pain, joint pain, abdominal pain, etc. Previous studies have described mixed infections with DENV/CHIKV, DENV/Plasmodium and CHIKV/Plasmodium [1, 3, 4, 6, 8]. A recent study reported concurrent infection with Plasmodium, DENV and CHIKV in a traveler from India [14]. Other recent studies from India have also reported such concurrent infections with all the three pathogens [15]. The present investigation describes an unusual case report of mixed infection with three mosquito transmitted pathogens (dengue and chikungunya viruses and Plasmodium vivax). This case was identified during monsoon season of 2016 which coincided with high rate of DENV and CHIKV infections from New Delhi, India [13]. Such large number of infections was reported probably due to rapid proliferation of mosquitoes because of high humidity and elevated temperature during the rainy season. We identified mixed infection with all these pathogens (dengue and chikungunya viruses and Plasmodium vivax) in another 3 year old patient [15]. We are acutely aware of lack of follow up/treatment data and absence of sequences information of two pathogens but the present study can be used for future reference of unique case reports.

Concurrent infections with multiple pathogens pose a challenge for the physicians for accurate diagnosis and treatment due to overlapping symptoms. Thus, although these mixed infections are indistinguishable from the mono infections clinically, the laboratory diagnosis is essential for the pathogen confirmation. It has been previously described that certain concurrent infections may be severe as compared to single infections [7, 12] while other showed no differences among the two [9]. The accurate identification of the pathogens is needed for appropriate treatment recommendation. Further, correlation of mixed infections with disease severity in case of mixed and isolated infections and their clinical outcome will provide insight into its holistic understanding and molecular mechanism. Therefore, the clinicians should be aware of such atypical cases which will further assist in appropriate management and treatment of arthropod mediated mixed infections. In addition, in depth analysis at the genetic and molecular level is envisaged to provide information on co-existence of such multiple pathogens in human host.

Electronic supplementary material

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