Bancroftian filariasis, caused by the W. bancrofti infection, covers about two-thirds of the global filarial population . This form of filariasis is considered to be an important public health issue in India . The disease is very ancient in the country as evident from the two oldest medical books of India namely the Susruta Samhita (6th century BC), and the Rug-vinischaya, also known as the Nidāna, written by the physician Madhava-kara (7th century AD) . The endemic map constitutes 20 states and six UTs in India, where 23 million people are suffering from the disease, 31 million are mf carriers, and about 553 million are at risk of infection . Previously, we have reported the prevalent status of bancroftian filariasis in the filarial endemic rural areas of the two districts, Bankura and Birbhum, in West Bengal, India .
Individuals suffering from bancroftian filariasis exhibit demonstrable clinical pathology that includes lymphedema, hydrocele, and elephantiasis . Progressive lymphatic damage and pathology caused by the filarial parasite is due to immense tissue alteration and immunomodulation, which also promotes secondary infection by bacteria and fungus [40, 41]. Previous researchers have reported bacterial and fungal co-infections in LF patients during the progression of elephantiasis [18, 19, 21]. However, the study of the diagnosis of fungal co-infection has been neglected to date.
Co-infection is a very important topic in relation to human health and still lacks a lot of epidemiological and experimental data. To date, there are very few researchers who have studied the co-infections associated with any form of filariasis. There has been a report of co-infection with filarial parasites and Mycobacteria or Plasmodium spp. in filarial patients, which indicated that immunomodulation and suppression of pro-inflammatory response are the principal reasons behind such co-infection . However, fungal co-infection with bancroftian filariasis has not been reported to date. Our experimental and epidemiological data revealed the occurrence and molecular identification of non-albicans Candida (P. guilliermondii) co-infection in a statistically significant number of individuals suffering from bancroftian filariasis.
The identification of P. guilliermondii-W. bancrofti co-infection in microfilaraemic individuals and its prevalence were studied using PCR-based molecular diagnosis.
The method of choice for molecular diagnosis of bancroftian filariasis is the PCR-based selective amplification of the Wolbachia (a filarial endosymbiont) specific gene (wsp int gene). Interestingly, during the study of the LF prevalence in the mentioned districts, in most of the cases (88.7%), we observed three intense bands of 630, 504, and 252 bp, along with the wsp amplicon (590 bp). After sequencing all the bands, we came to the conclusion that, apart from wsp, the nonspecifically amplified bands belonged to a fungus i.e. P. guilliermondii (see Table 2). All the DNA sequences obtained from the bands shared significant similarities (up to 90%) with P. guilliermondii, which is synonymous to C. guilliermondii or M. guilliermondii. Existence/appearance of such fungal metagenomic DNA in a number of occasions had prompted us to identify and investigate the occurrence of this organism in filarial patients. Amplification and sequencing of the D1/D2 region of the 26S rRNA gene from fungal DNA, followed by a similarity search using BLASTn, confirmed that the organism is P. guilliermondii. Molecular identification based on D1/D2 region of the 26S/28S rRNA gene is a reliable and robust means for identifying clinically relevant yeast isolates . This PCR amplification based identification is specific, sensitive and does not involve complex and expensive equipment . Sequencing of the internal transcribed regions (ITS) of the nuclear rRNA gene had especially been used to identify and discriminate between 40 species of medically important yeasts . These regions have evolved slowly and show high degrees of conservation among fungi, and are thus used for molecular identification and to study the phylogenetic relationships among the isolates . In this study, the phylogenetic tree based on D1/D2 region of 26S rDNA sequence of P. guilliermondii and its close neighbors presented a comprehensive view of their distribution, as well as their evolutionary relationship (see Figure 1D). P. guilliermondii was placed closely with C. albicans in the tree. However, the position of P. guilliermondii-C. albicans was supported by a low bootstrap value (BV% 41). In addition, selective amplification of the RPS0 gene with P. guilliermondii specific PCR primers further supported our finding. An amplicon of 620 bp (approximately) indicated the pathogenicity of the isolated species, and this finding corroborates with a previous report . Screening of pathogenicity through a selective amplification of RPS0 has been reported as an efficient approach for the determination of pathogenicity of yeast isolates from a clinical specimen . Identification and differentiation of pathogenicity of a number of Candida spp., including P. guilliermondii, by selective amplification of RPS0 has been reported previously .
The antifungal susceptibility profile of pathogenic yeast varies greatly and the widespread use of antifungals might have contributed to the alteration in the species distribution through antibiotic resistance . According to our study, P. guilliermondii tends to be resistant to common antifungals (mostly azoles). Therefore, treatment with an appropriate antifungal is required to improve survival rates in these patients. Previously, we have reported effective combinatorial chemotherapy using doxycycline (an antibacterial antibiotic) and albendazole (an anthelmintic) for the control of bancroftian filariasis in India . However, neither of these drugs could eliminate this secondary infection. In vitro susceptibility of the isolate suggested that nystatin or cycloheximide could be the drugs to treat this fungal co-infection in filarial patients. Extensive in vivo studies and clinical trials are welcome to optimize the dose and duration needed to treat patients.
The high prevalence of P. guilliermondii in W. bancrofti infected individuals, inferred through first-time experimental findings, establishes the occurrence of the yeast-filarial parasite co-infection in India. Therefore, the study provides the platform to investigate the role of such co-infection in pathology and disease progression in LF.