A 23-year-old male had unprotected sexual intercourse with a commercial sex worker. Two weeks later, he developed a painless, indurated ulcer on the glans which exuded clear serum on pressure. Inguinal lymph nodes in both groins were enlarged and not tender. The most appropriate diagnostic test is –
Gram's stain of ulcer discharge
Darkfield microscopy of ulcer discharge
Giemsa stain of lymph node aspirate
ELISA for HIV infection
It is very easy to diagnose an STD by a simple stepwise approach

​Step -1

The first step is to see whether the ulcer is painful or painless.
Painless ulcer     →   LGV, donovanosis, chancre.
Painful ulcers    → HSV, chancroid.

Step - 2

For painless ulcer, see whether lymphadenopathy is present or not:-
Lymphadenopathy (+)nt → LGV, chance
No lymphadenopathy   →  Donovanosis
If lymphadenopathy is (+)nt, see whether it is painful or not:-
Painful lymphadenopathy → LGV
Painless lymphadenopathy → Chancre
	​

Now see the question
Step -1
An ulcer is a painless → Chancre, LGV or Donovanosis.

Step - 2
Inguinal lymphadenopathy (+)nt → Chancre or LGV (Donovanosis is ruled out).
Lymphadenopathy is non-tender → Diagnosis is chancre (LGV is ruled out).

As the clinical diagnosis is chancre (Primary syphilis), Lab. diagnosis can be made by darkfield microscopy of ulcer discharge