This study of aetiology of undifferentiated fever in rural India using standard diagnostic tests, revealed a high prevalence of malaria and dengue. However, there was a strikingly high prevalence of overlap of case definitions. An overlap with one or more other case-definition was found for all diagnosed diseases, ranging from 33% (bacteraemia) to 57% (chikungunya) (Table 3). The highest frequency of overlap was found in chikungunya where dengue was simultaneously diagnosed in 26% (25/98), followed by leptospirosis, where scrub typhus was found in 24% (28/116).
Cross reactivity, or background positivity due to previous infections, are well known limitations of serological tests, and fourfold rise of titer in convalescence samples or a high acute phase titer is recommended to confirm a diagnosis. Convalescent samples were not available in this study, reflecting a real life situation in resource poor settings where tests for follow up in recovered patients are usually not collected.
Detecting a pathogen directly by PCR or culture is more specific than indirect diagnosis by antibody detection, and the diagnoses of malaria and bacteraemia are therefore likely to be more specific than leptospirosis, scrub typhus, chikungunya and dengue in this study. Positive serological tests for dengue, leptospirosis, scrub typhus and chikungunya were common also in patients with malaria and bacteraemia (Table 4), suggesting low specificity of the serological tests. Although coinfections are possible, it is more likely that multiple fulfilled case definitions in a high proportion of patients are due to cross reactivity and background positivity, reflecting that the diseases detected by serology are endemic in the area, rather than high prevalence of coinfections. The findings in the present study emphasises the importance of interpreting diagnostic tests in a clinical context together with symptoms, clinical findings and biochemical tests.
Malaria
Malaria parasites were detected by PCR in 17% (268/1564) among patients included, and among these 54% (145/268) were P. falciparum, as reported previously [7]. Due to high sensitivity of malaria PCR compared to microscopy and RDT, some PCR positive cases may potentially have had asymptomatic low parasitemia controlled by immunity, or recently been treated for malaria, and their fever caused by another infection [7, 9]. As reported previously, microscopy had low sensitivity (29%, 66/228) but high specificity (98%, 918/940) compared to PCR, and a very strict case definition of clinical malaria as cause of acute fever can be defined as a positive microscopy confirmed by PCR [7]. The prevalence of malaria by microscopy confirmed by PCR was 6% (66/1168).
Bacteraemia
Blood stream infection with pathogenic bacteria was diagnosed in 8% (124/1564), and among these Salmonella typhi or S. paratyphi were found in 35% (44/124), reflecting the high prevalence of enteric fever in India. Enteric fever is closely associated with poor sanitation, lack of safe water supply and treatment failures due to antimicrobial resistance and is still reported as the most common blood stream infection in India and in South Asia [10,11,12,13]. The second most common microbe identified was S. aureus (19% 24/124), followed by E.-coli (9%, 11/124) and S. pneumoniae (6%, 7/124).
Dengue
Dengue and severe dengue because of immune enhancement due to a previous infection with another serotype is an increasing problem in India [14, 15]. India is estimated to contribute 34% (33/96 million) of the total global burden of dengue [16], with increasing incidence both of dengue and outbreaks of severe dengue [17, 18]. The risk of severe dengue is high, as more than 25% of the population in Delhi has been reported to have had a past infection [17, 19]. In line with the high prevalence reported in previous studies, dengue was found in as much as 16% (244/1564) in the present study, highest in the sites in South- and West India.
Rapid tests combining detection of non-structural protein 1 (NS1) antigen and IgM/IgG are used in routine diagnostics, as they have high sensitivity both during the viremic early phase of infection when NS1 is produced and after more than five days when IgM can be detected [20]. IgM capture ELISA (MAC ELISA) is used as reference method, but is less sensitive than NS1Ag until day five of infection. Case definition used in the present study was therefore a positive test with RDT and/or ELISA, in order not to miss out early infections detected by NS1Ag. However, background positivity is a potential limitation since MAC ELISA can be positive for several months after infection [21]. Although NS1 antigen is less prone to give cross reactivity than IgM antibodies, combination tests have shown some false positive reactions in non-dengue infections, most commonly in chikungunya [20, 21].
Chikungunya
A large outbreak of Chikungunya was reported in Ahmedabad in India in 2006 [22]. Sharing the same vector, chikungunya is likely to occur during dengue outbreaks, and in a study during a dengue outbreak in Delhi in 2010, 10% (66/666) positive chikungunya cases were diagnosed among dengue IgM negative fever patients [23]. Sporadic outbreaks of chikungunya has been reported in India since 1963, in 2006 affecting 13 states with 1.4 million suspected cases [23], with high numbers in Andhra Pradesh, Tamil Nadu and Maharashtra. This supports the finding in the present study of highest prevalence of chikungunya in Anantapur (Andhra Pradesh), Oddanchatram and Ambur (Tamil Nadu) and Ratnagiri (Maharashtra).
Leptospirosis
Leptospirosis is transmitted by urine from infected animals (rats, cattle, pigs) and is endemic particularly in the Andaman and Nicobar group of islands (“Andaman haemorrhagic fever”) [24]. In AUF studies from South- and Northern India, leptospirosis was reported in 3% and 0.1% respectively [11, 12]. In the present study leptospirosis was found in 7% (116/1564), and cases were identified at all study sites.
Culturing Leptospira is unreliable, and the gold standard is therefore serology confirmed by MAT. MAT detects IgM and IgG antibodies against a pool of live antigens from different Leptospira serovars. A MAT titer >100 is considered positive, but a fourfold rise in convalescence titer or a high single acute phase titer (>200–1600 depending on endemicity) supports the diagnosis [25]. Following acute leptospirosis, both IgM ELISA and MAT remain positive for several years after infection, with duration differing between serogroups [26]. In one prospective study from Barbados, positive MAT was found up to 11 years after infection, with highest prevalence after serogroup Autumnalis infection where 20% had MAT titer >800 after four years [26]. In the present study Autumnalis was found in 7%. Leptospira serovar prevalence and distribution in this study has been reported previously [27]. Leptospira IgM ELISA has been reported positive in 40% and 5% one and six years after infection respectively [26]. Discrimination between acute and previous infection in the present study is limited by lack of convalescent samples, and a low MAT cut-off titer of 100. However, high prevalence of antibodies in all study sites suggests that the disease is endemic in the areas.
Scrub typhus
Scrub typhus is transmitted by mites who live on rats. The disease is, similar to leptospirosis, associated with agricultural work and rural dwelling [28]. Two studies have reported prevalence of 14% and 47% among hospitalized febrile patients in North- and South India respectively [11, 12]. The disease is endemic in various parts of India, but underreported [1, 29,30,31,32,33,34,35]. In the present study, scrub typhus was found in 10% (159/1564), and the disease was identified at all study sites.
Serology confirmed by IFA, ideally confirmed by rise of titer in convalescent samples and/or by cut-off values based on endemicity, remains the mainstay of diagnostics since isolation of the bacteria is not possible and PCR from blood has low sensitivity [36]. Sensitivity of IFA may be influenced by antigen variation. Usually antigens from three serotypes (Karp, Kato and Gilliam) are used, while additional antigens may be present in different areas [30, 36]. In the present study, discrimination between previous scrub typhus and acute infection is limited by the lack of convalescent samples. Also an optic density (OD) value of 0.5 may be in the lower range and, thereby, in some cases reflect background positivity.
Potential coinfections
Although background positivity or cross reactivity in serology, and potential subclinical infections in malaria, may have given positive test results in some cases, some of the overlapping aetiologies have probably been due to true clinically relevant coinfections.
Coinfections could occur principally by two different mechanisms; by contracting multiple infections at the same time, or increased pathogenicity of a simultaneous subclinical infection due to immune reactions.
The risk of bacterial sepsis is increased in severe malaria, through immune mediated barrier dysfunction in the gut and bacterial translocation, as well as IL-10 mediated decreased control of bacteraemia [37, 38]. In clinical studies, invasive infection, frequently with Salmonella spp. or other Gram-negatives, are found both in P. falciparum and P. vivax malaria [39, 40], which supports the finding of as much as 9% (25/268) bacteraemia among malaria patients in the present study. On the other hand, asymptomatic malaria controlled by immunity may obscure correct diagnosis of bacterial sepsis [41,42,43], and an undefined proportion of the malaria positive patients among those with bacteraemia may have had subclinical malaria in the present study.
In a study among Thai rice farmers with leptospirosis diagnosed with 4-fold rise in titer or a single high titer, as many as nine among 22 patients had coinfection with scrub typhus confirmed by serology and eschar or clinical characteristics [44]. Although a very high overlap between positive tests for scrub typhus and leptospirosis in the present study suggests background positivity or cross reactivity, a proportion of the patients may have had coinfections taking into consideration the similar exposure risk.
True coinfections with malaria and scrub typhus, diagnosed by clinical characteristics and eschar or PCR, have also been reported in India [45,46,47]. However, the high level of positive scrub typhus serology in single samples found in other Indian studies [12, 48], raises the same question as in the present study where 10% (27/268) of malaria cases had positive scrub typhus serology, do the results reflect true coinfections, or cross reactivity or background positivity?
In the mosquito borne infections dengue, malaria and chikungunya, outbreaks occur during rainy seasons and although the specific vector is different for malaria, coinfections are not unlikely. This was shown in a study from India during a dengue outbreak, where 7% (27/367) of dengue cases had coinfection with malaria [49]. As much as 22% (58/268) of malaria cases had positive dengue tests in the present study (Tables 3 and 4).
A high level of coinfections with dengue and chikungunya was shown during a dengue outbreak in Delhi in 2006 using PCR as the method for detection. Among 17 chikungunya positive patients, six were co-infected with dengue virus [50]. Ten percent coinfection was found in a study from Mumbai [51]. Dengue and chikungunya virus share a common mosquito vector, the daytime biting Aedes aegypti and A. albopictus, and are present in similar geographical regions. In the present study, dengue and chikungunya both had high prevalence in Anantapur, supporting the notion that coinfections as well as cross reactivity could explain some overlap between dengue and chikungunya.